Selective inhibitors of rock protein kinase and uses thereof

ABSTRACT

Described herein are compounds that are useful as ROCK inhibitors. These compounds, and pharmaceutically acceptable compositions thereof, are useful for treating or lessening the severity of a variety of disorders, including cardiovascular, inflammatory, neurological, or proliferative diseases or disorders.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to heteroaryl compounds that are proteinkinase inhibitors, compositions containing such compounds, and methodsfor their use. The compounds and compositions of the invention areuseful for treating cardiovascular, inflammatory, neurological, orproliferative diseases or disorders.

BACKGROUND OF THE INVENTION

One kinase family of interest is Rho-associated coiled-coil formingprotein serine/threonine kinase (ROCK), which is believed to be aneffector of Ras-related small GTPase Rho. The ROCK family includesp160ROCK (ROCK-1), ROKα/Rho-kinase/ROCK-II, protein kinase PKN, citron,and citron kinase. The ROCK family of kinases have been shown to beinvolved in a variety of functions including Rho-induced formation ofactin stress fibers and focal adhesions and in down-regulation of myosinphosphatase, platelet activation, aortic smooth muscle contraction byvarious stimuli, thrombin-induced responses of aortic smooth musclecells, hypertrophy of cardiomyocytes, bronchial smooth musclecontraction, smooth muscle contraction and cytoskeletal reorganizationof non-muscle cells, activation of volume-regulated anion channels,neurite retraction, neutrophil chemotaxis, wound healing, tumor invasionand cell transformation. More specifically, ROCK has been implicated invarious diseases and disorders including hypertension, cerebralvasospasm, coronary vasospasm, bronchial asthma, preterm labor, erectiledysfunction, glaucoma, vascular smooth muscle cell proliferation,myocardial hypertrophy, malignoma, ischemia/reperfusion-induced injury,endothelial dysfunction, Crohn's Disease and colitis, neurite outgrowth,Raynaud's Disease, angina, Alzheimer's disease, benign prostatichyperplasia and atherosclerosis.

U.S. Patent Application Publication No. 20040122016 describes severalpyridylthiazole and pyridylthiofuran inhibitors of ROCK. The developmentof other inhibitors of ROCK kinase would be useful for the treatment ofdiseases and disorders associated with the ROCK kinase pathway.

SUMMARY OF THE INVENTION

The present invention features compounds having the formula:

or a pharmaceutically acceptable salt or prodrug thereof, where R¹, R²,R³, X¹, and A¹ are as defined herein. These compounds andpharmaceutically acceptable compositions or prodrugs thereof are usefulfor treating or lessening the severity of a variety of ROCK-mediateddiseases or disorders disorders, especially cardiovascular,inflammatory, neurological, or proliferative diseases or disorders. Inaddition, these compounds are useful because they inhibit cytochromeP450 only at high concentrations.

DETAILED DESCRIPTION OF THE INVENTION

Compounds of the Invention

As used herein, the following definitions shall apply unless otherwiseindicated. As described herein, compounds or classes of compounds of theinvention may optionally be substituted with one or more substituents,such as, for example, one, two, three, four, or five substituents. Itwill be appreciated that the phrase “optionally substituted” is usedinterchangeably with the phrase “substituted or unsubstituted.” Ingeneral, the term “substituted,” whether preceded by the term“optionally” or not, refers to the replacement of one or more hydrogenradicals in a given structure with the radical of a specifiedsubstituent. Unless otherwise indicated, an optionally substituted groupmay have a substituent at each substitutable position of the group. Whenmore than one position in a given structure can be substituted with morethan one substituent selected from a specified group, the substituentmay be either the same or different at each position.

As described herein, when the term “optionally substituted” precedes alist, said term refers to all of the subsequent substitutable groups inthat list. For example, if X is halogen; optionally substituted C₁₋₃alkyl or phenyl; X may be either optionally substituted alkyl oroptionally substituted phenyl. Likewise, if the term “optionallysubstituted” follows a list, said term also refers to all of thesubstitutable groups in the prior list unless otherwise indicated. Forexample: if X is halogen, C₁₋₃ alkyl, or phenyl, wherein X is optionallysubstituted by J^(X), then both C₁₋₃ alkyl and phenyl may be optionallysubstituted by J^(X). As is apparent to one having ordinary skill in theart, groups such as hydrogen, halogen, NO₂, CN, NH₂, OH, or OCF₃ wouldnot be included because they are not substitutable groups. If asubstituent radical or structure is not identified or defined as“optionally substituted,” the substituent radical or structure isunsubstituted.

Occasionally, the location of aryl, heterocyclyl, or heteroaryl ringsubstitutions will be defined by position. In such examples, unlessotherwise indicated, the 1-position of the ring is that position that isbonded to the remainder of the molecule. The other positions arenumbered according to priority designations defined by standard IUPACnomenclature.

Combinations of substituents envisioned by this invention are preferablythose that result in the formation of stable or chemically feasiblecompounds. The term “stable,” as used herein, refers to compounds thatare not substantially altered when subjected to conditions to allow fortheir production, detection, and, preferably, their recovery,purification, and use for one to five of the purposes disclosed herein.In some embodiments, a stable compound or chemically feasible compoundis one that is not substantially altered when kept at a temperature of40° C. or less, in the absence of moisture or other chemically reactiveconditions, for at least a week.

The term “aliphatic” or “aliphatic group,” as used herein, means astraight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one to five units of unsaturation. Unless otherwise specified,aliphatic groups contain 1-20 carbon atoms. In some embodiments,aliphatic groups contain 1-10 carbon atoms. In other embodiments,aliphatic groups contain 1-8 carbon atoms. In still other embodiments,aliphatic groups contain 1-6 carbon atoms, and in yet other embodiments,aliphatic groups contain 1-4 carbon atoms. Suitable aliphatic groupsinclude, but are not limited to, linear or branched, substituted orunsubstituted alkyl, alkenyl, or alkynyl groups. Further examples ofaliphatic groups include methyl, ethyl, propyl, butyl, isopropyl,isobutyl, vinyl, and sec-butyl. The terms “alkyl” and the prefix “alk-,”as used herein, are inclusive of both straight chain and branchedsaturated carbon chain. The term “alkylene,” as used herein, representsa saturated divalent hydrocarbon group derived from a straight orbranched chain saturated hydrocarbon by the removal of two hydrogenatoms, and is exemplified by methylene, ethylene, isopropylene and thelike. The term “alkenyl,” as used herein, represents monovalent straightor branched chain hydrocarbon group containing one to five carbon-carbondouble bonds. The term “alkynyl,” as used herein, represents amonovalent straight or branched chain hydrocarbon group containing oneto five carbon-carbon triple bonds.

The term “alkylidene chain” refers to a straight or branched carbonchain that may be fully saturated or have one or more units ofunsaturation and has two points of connection to the rest of themolecule.

The term “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refers to amonocyclic C₃-C₈ hydrocarbon or bicyclic C₈-C₁₂ hydrocarbon that iscompletely saturated or that contains one to five units of unsaturation,but which is not aromatic, that has a single point of attachment to therest of the molecule, and wherein any individual ring in said bicyclicring system has 3-7 members. Suitable cycloaliphatic groups include, butare not limited to, cycloalkyl, cycloalkenyl, and cycloalkynyl. Furtherexamples of aliphatic groups include cyclopentyl, cyclopentenyl,cyclohexyl, cyclohexenyl, cycloheptyl, and cycloheptenyl.

The term “heterocycle” “heterocyclyl” or “heterocyclic” as used hereinrefers to a monocyclic, bicyclic, or tricyclic ring system in which oneto five ring members are an independently selected heteroatom and thatis completely saturated or that contains one to five units ofunsaturation, but which is not aromatic, that has a single point ofattachment to the rest of the molecule. In some embodiments, the“heterocycle,” “heterocyclyl,” or “heterocyclic” group has three tofourteen ring members in which one to five ring members is a heteroatomindependently selected from oxygen, sulfur, nitrogen, or phosphorus, andeach ring in the system contains 3 to 8 ring members.

Examples of heterocyclic rings include, but are not limited to, thefollowing monocycles: 2-tetrahydrofuranyl, 3-tetrahydrofuranyl,2-tetrahydrothiophenyl, 3-tetrahydrothiophenyl, 2-morpholino,3-morpholino, 4-morpholino, 2-thiomorpholino, 3-thiomorpholino,4-thiomorpholino, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl,1-tetrahydropiperazinyl, 2-tetrahydropiperazinyl,3-tetrahydropiperazinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl,1-pyrazolinyl, 3-pyrazolinyl, 4-pyrazolinyl, 5-pyrazolinyl,1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl,2-thiazolidinyl, 3-thiazolidinyl, 4-thiazolidinyl, 1-imidazolidinyl,2-imidazolidinyl, 4-imidazolidinyl, 5-imidazolidinyl; and the followingbicycles: 3-1H-benzimidazol-2-one, 3-(1-alkyl)-benzimidazol-2-one,indolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, benzothiolane,benzodithiane, and 1,3-dihydro-imidazol-2-one.

The term “heteroatom” means one to five of oxygen, sulfur, nitrogen,phosphorus, or silicon, including any oxidized form of nitrogen, sulfur,or phosphorus; the quaternized form of any basic nitrogen; or asubstitutable nitrogen of a heterocyclic ring, for example N (as in3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺ (as inN-substituted pyrrolidinyl).

The term “unsaturated,” as used herein, means that a moiety has one tofive units of unsaturation.

The term “alkoxy,” or “thioalkyl,” as used herein, refers to an alkylgroup, as previously defined, attached to the principal carbon chainthrough an oxygen (“alkoxy”) or sulfur (“thioalkyl”) atom.

The terms “haloalkyl,” “haloalkenyl,” and “haloalkoxy” means alkyl,alkenyl, or alkoxy, as the case may be, substituted with one to fivehalogen atoms. The term “halogen” means F, Cl, Br, or I.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic,bicyclic, and tricyclic carbocyclic ring systems having a total of sixto fourteen ring members, wherein at least one ring in the system isaromatic, wherein each ring in the system contains 3 to 7 ring membersand that has a single point of attachment to the rest of the molecule.The term “aryl” may be used interchangeably with the term “aryl ring.”Examples of aryl rings would include phenyl, naphthyl, and anthracene.

The term “heteroaryl,” used alone or as part of a larger moiety as in“heteroaralkyl,” or “heteroarylalkoxy,” refers to monocyclic, bicyclic,and tricyclic ring systems having a total of five to fourteen ringmembers, wherein at least one ring in the system is aromatic, at leastone ring in the system contains one to five heteroatoms, wherein eachring in the system contains 3 to 7 ring members and that has a singlepoint of attachment to the rest of the molecule. The term “heteroaryl”may be used interchangeably with the term “heteroaryl ring” or the term“heteroaromatic.”

Further examples of heteroaryl rings include the following monocycles:2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl,5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxazolyl,4-oxazolyl, 5-oxazolyl, N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl,pyridazinyl (e.g., 3-pyridazinyl), 2-thiazolyl, 4-thiazolyl,5-thiazolyl, tetrazolyl (e.g., 5-tetrazolyl), triazolyl (e.g.,2-triazolyl and 5-triazolyl), 2-thienyl, 3-thienyl, pyrazolyl (e.g.,2-pyrazolyl), isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,3-thiadiazolyl,1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, pyrazinyl, 1,3,5-triazinyl, andthe following bicycles: benzimidazolyl, benzofuryl, benzothiophenyl,indolyl (e.g., 2-indolyl), purinyl, quinolinyl (e.g., 2-quinolinyl,3-quinolinyl, 4-quinolinyl), and isoquinolinyl (e.g., 1-isoquinolinyl,3-isoquinolinyl, or 4-isoquinolinyl).

In some embodiments, an aryl (including aralkyl, aralkoxy, aryloxyalkyl,and the like) or heteroaryl (including heteroaralkyl, heteroarylalkoxy,and the like) group may contain one to five substituents. Suitablesubstituents on the unsaturated carbon atom of an aryl or heteroarylgroup include: halogen; —R^(o); —OR^(o); —SR^(o); 1,2-methylenedioxy;1,2-ethylenedioxy; phenyl (Ph), optionally substituted with R^(o);—O(Ph), optionally substituted with R^(o); —(CH₂)₁₋₂(Ph), optionallysubstituted with R^(o); —CH═CH(Ph), optionally substituted with R^(o);—NO₂; —CN; —N(R^(o))₂; —NR^(o)C(O)R^(o); —NR^(o)C(S)R^(o);—NR^(o)C(O)N(R^(o))₂; —NR^(o)C(S)N(R^(o))₂; —NR^(o)C(O)OR^(o);—NR^(o)NR^(o)C(O)R^(o); —NR^(o)NR^(o)C(O)N(R^(o))₂;—NR^(o)NR^(o)C(O)OR^(o); —C(O)C(O)R^(o); —C(O)CH₂C(O)R^(o); —C(O)OR^(o);—C(O)R^(o); —C(S)R^(o); —C(O)N(R^(o))₂; —C(S)N(R^(o))₂; −B(OR^(o))₂;—OC(O)N(R^(o))₂; —OC(O)R^(o); —C(O)N(OR^(o))R^(o); —C(NOR^(o))R^(o);—S(O)₂R^(o); —S(O)₂OR^(o); —S(O)₂N(R^(o))₂; —S(O)R^(o);—NR^(o)S(O)₂N(R^(o))₂; —NR^(o)S(O)₂R^(o); —N(OR^(o))R^(o);—C(═NH)—N(R^(o))₂; —(CH₂)₀₋₂NHC(O)R^(o); −L-R^(o); −L-N(R^(o))₂;-L-SR^(o); −L-OR^(o); −L-(C₃₋₁₀ cycloaliphatic), -L—(C₆₋₁₀ aryl),-L—(5-10 membered heteroaryl), -L-(5-10 membered heterocyclyl), oxo,C₁₋₄haloalkoxy, C₁₋₄haloalkyl, −L-NO₂, −L-CN, -L-OH, −L-CF₃; or twosubstituents, on the same carbon or on different carbons, together withthe carbon or intervening carbons to which they are bound, form a 5-7membered saturated, unsaturated, or partially saturated ring, wherein Lis a C₁₋₆ alkylene group in which up to three methylene units arereplaced by —NH—, —NR^(o)—, —O—, —S—, —C(O)O—, —OC(O)—, —C(O)C(O)—,—C(O)—, —C(O)NH—, —C(O)NR^(o)—, —C(═N—CN), —NHC(O)—, —NR^(o)C(O)—,—NHC(O)O—, —NR^(o)C(O)O—, —S(O)₂NH—, —S(O)₂NR^(o)—, —NHS(O)₂—,—NR^(o)S(O)₂—, —NHC(O)NH—, —NR^(o)C(O)NH—, —NHC(O)NR^(o),—NR^(o)C(O)NR^(o), —OC(O)NH—, —OC(O)NR^(o)—, —NHS(O)₂NH—,—NR^(o)S(O)₂NH—, —NHS(O)₂NR^(o)—, —NR^(o)S(O)₂NR^(o)—, —S(O)—, or—S(O)₂—, and wherein each occurrence of R^(o) is independently selectedfrom hydrogen, optionally substituted C₁₋₆ aliphatic, an unsubstituted5- to 6-membered heteroaryl or heterocyclic ring, phenyl, or —CH₂(Ph),or, two independent occurrences of R^(o), on the same substituent ordifferent substituents, taken together with the atom(s) to which eachR^(o) group is bound, form a 5-8-membered heterocyclyl, aryl, orheteroaryl ring or a 3- to 8-membered cycloalkyl ring, wherein saidheteroaryl or heterocyclyl ring has 1 to 3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. Optional substituents on thealiphatic group of R^(o) are selected from NH₂, NH(C₁₋₄ aliphatic),N(C₁₋₄ aliphatic)₂, halogen, C₁₋₄ aliphatic, OH, O(C₁₋₄ aliphatic), NO₂,CN, C(O)OH, C(O)O(C₁₋₄ aliphatic), O(halo(C₁₋₄ aliphatic)), or halo(C₁₋₄aliphatic), wherein each of the foregoing C₁₋₄ aliphatic groups of R^(o)is unsubstituted.

In some embodiments, an aliphatic or heteroaliphatic group, or anon-aromatic heterocyclic ring may contain one to five substituents.Suitable substituents on the saturated carbon of an aliphatic orheteroaliphatic group, or of a non-aromatic heterocyclic ring areselected from those listed above for the unsaturated carbon of an arylor heteroaryl group and additionally include the following: ═O, ═S,═NNHR*, ═NN(R*)₂, ═NNHC(O)R*, ═NNHC(O)O(alkyl), ═NNHS(O)₂(alkyl), or═NR*, where each R* is independently selected from hydrogen or anoptionally substituted C₁₋₆ aliphatic. Optional substituents on thealiphatic group of R* are selected from NH₂, NH(C₁₋₄ aliphatic), N(C₁₋₄aliphatic)₂, halogen, C₁₋₄ aliphatic, OH, O(C₁₋₄ aliphatic), NO₂, CN,C(O)OH, C(O)O(C₁₋₄ aliphatic), O(halo-C₁₋₄ aliphatic), and halo(C₁₋₄aliphatic), where each of the foregoing C₁₋₄ aliphatic groups of R* isunsubstituted.

In some embodiments, optional substituents on the nitrogen of anon-aromatic heterocyclic ring include —R⁺, —N(R⁺)₂, —C(O)R⁺, —C(O)OR⁺,—C(O)C(O)R⁺, —C(O)CH₂C(O)R⁺, —S(O)₂R⁺, —S(O)₂N(R⁺)₂, —C(═S)N(R⁺)₂,—C(═NH)—N(R⁺)₂, or —NR₊S(O)₂R⁺; wherein R₊ is hydrogen, an optionallysubstituted C₁₋₆ aliphatic, optionally substituted phenyl, optionallysubstituted —O(Ph), optionally substituted —CH₂(Ph), optionallysubstituted —(CH₂)₁₋₂(Ph); optionally substituted —CH═CH(Ph); or anunsubstituted 5-6 membered heteroaryl or heterocyclic ring having one tofour heteroatoms independently selected from oxygen, nitrogen, orsulfur, or, two independent occurrences of R⁺, on the same substituentor different substituents, taken together with the atom(s) to which eachR⁺ group is bound, form a 5-8-membered heterocyclyl, aryl, or heteroarylring or a 3-8-membered cycloalkyl ring, wherein said heteroaryl orheterocyclyl ring has 1-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. Optional substituents on the aliphaticgroup or the phenyl ring of R⁺ are selected from NH₂, NH(C₁₋₄aliphatic), N(C₁₋₄ aliphatic)₂, halogen, C₁₋₄ aliphatic, OH, O(C₁₋₄aliphatic), NO₂, CN, C(O)OH, C(O)O(C₁₋₄ aliphatic), O(halo(C₁₋₄aliphatic)), or halo(C₁₋₄ aliphatic), wherein each of the foregoing C₁₋₄aliphatic groups of R⁺ is unsubstituted.

As detailed above, in some embodiments, two independent occurrences ofR^(o) (or R⁺, or any other variable similarly defined herein), may betaken together with the atom(s) to which each variable is bound to forma 5-8-membered heterocyclyl, aryl, or heteroaryl ring or a 3-8-memberedcycloalkyl ring. Exemplary rings that are formed when two independentoccurrences of R^(o) (or R⁺, or any other variable similarly definedherein) are taken together with the atom(s) to which each variable isbound include, but are not limited to the following: a) two independentoccurrences of R^(o) (or R⁺, or any other variable similarly definedherein) that are bound to the same atom and are taken together with thatatom to form a ring, for example, N(R^(o))₂, where both occurrences ofR^(o) are taken together with the nitrogen atom to form apiperidin-1-yl, piperazin-1-yl, or morpholin-4-yl group; and b) twoindependent occurrences of R^(o) (or R⁺, or any other variable similarlydefined herein) that are bound to different atoms and are taken togetherwith both of those atoms to form a ring, for example where a phenylgroup is substituted with two occurrences of OR^(o)

these two occurrences of R^(o) are taken together with the oxygen atomsto which they are bound to form a fused 6-membered oxygen containingring:

It will be appreciated that a variety of other rings can be formed whentwo independent occurrences of R^(o) (or R⁺, or any other variablesimilarly defined herein) are taken together with the atom(s) to whicheach variable is bound and that the examples detailed above are notintended to be limiting.

In some embodiments, an alkyl or aliphatic chain can be optionallyinterrupted with another atom or group. This means that a methylene unitof the alkyl or aliphatic chain is optionally replaced with said otheratom or group. Examples of such atoms or groups would include, but arenot limited to, —NR—, —O—, —S—, —C(O)O—, —OC(O)—, —C(O)C(O)—, —C(O)—,—C(O)NR—, —C(═N—CN), —NRC(O)—, —NRC(O)O—, —S(O)₂NR—, —NRS(O)₂—,—NRC(O)NR—, —OC(O)NR—, —NRS(O)₂NR—, —S(O)—, or —S(O)₂—, wherein R isdefined herein. Unless otherwise specified, the optional replacementsform a chemically stable compound. Optional interruptions can occur bothwithin the chain and at either end of the chain; i.e. both at the pointof attachment and/or also at the terminal end. Two optional replacementscan also be adjacent to each other within a chain so long as it resultsin a chemically stable compound. Unless otherwise specified, if thereplacement or interruption occurs at the terminal end, the replacementatom is bound to a hydrogen on the terminal end. For example, if—CH₂CH₂CH₃ were optionally interrupted with —O—, the resulting compoundcould be —OCH₂CH₃, —CH₂OCH₃, or —CH₂CH₂OH.

As described herein, a bond drawn from a substituent to the center ofone ring within a multiple-ring system (as shown below) representssubstitution of the substituent at any substitutable position in any ofthe rings within the multiple ring system. For example, Figure arepresents possible substitution in any of the positions shown in Figureb.

This also applies to multiple ring systems fused to optional ringsystems (which would be represented by dotted lines). For example, inFigure c, X is an optional substituent both for ring A and ring B.

If, however, two rings in a multiple ring system each have differentsubstituents drawn from the center of each ring, then, unless otherwisespecified, each substituent only represents substitution on the ring towhich it is attached. For example, in Figure d, Y is an optionallysubstituent for ring A only, and X is an optional substituent for ring Bonly.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, (Z) and (E) double bondisomers, and (Z) and (E) conformational isomers. Therefore, singlestereochemical isomers as well as enantiomeric, diastereomeric, andgeometric (or conformational) mixtures of the present compounds arewithin the scope of the invention.

The term “protecting group,” as used herein, represent those groupsintended to protect a functional group, such as, for example, analcohol, amine, carboxyl, carbonyl, etc., against undesirable reactionsduring synthetic procedures. Commonly used protecting groups aredisclosed in Greene and Wuts, Protective Groups In Organic Synthesis,3^(rd) Edition (John Wiley & Sons, New York, 1999), which isincorporated herein by reference. Examples of nitrogen protecting groupsinclude acyl, aroyl, or carbamyl groups such as formyl, acetyl,propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl,trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl,α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl,4-nitrobenzoyl and chiral auxiliaries such as protected or unprotectedD, L or D, L-amino acids such as alanine, leucine, phenylalanine and thelike; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and thelike; carbamate forming groups such as benzyloxycarbonyl,4-chlorobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,4-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,4-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxy carbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and thelike, arylalkyl groups such as benzyl, triphenylmethyl, benzyloxymethyland the like and silyl groups such as trimethylsilyl and the like.Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl(Boc)and benzyloxycarbonyl(Cbz).

The term “prodrug,” as used herein, represents a compound that istransformed in vivo into a compound of formula I, or a compound listedin Table 1. Such a transformation can be affected, for example, byhydrolysis in blood or enzymatic transformation of the prodrug form tothe parent form in blood or tissue. Prodrugs of the compounds of theinvention may be, for example, esters. Esters that may be utilized asprodrugs in the present invention are phenyl esters, aliphatic (C₁-C₂₄)esters, acyloxymethyl esters, carbonates, carbamates, and amino acidesters. For example, a compound of the invention that contains an OHgroup may be acylated at this position in its prodrug form. Otherprodrug forms include phosphates, such as, for example those phosphatesresulting from the phosphonation of an OH group on the parent compound.A thorough discussion of prodrugs is provided in T. Higuchi and V.Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S.Symposium Series, Edward B. Roche, ed., Bioreversible Carriers in DrugDesign, American Pharmaceutical Association and Pergamon Press, 1987,and Judkins et al., Synthetic Communications 26(23):4351-4367, 1996,each of which is incorporated herein by reference.

Unless otherwise stated, all tautomeric forms of the compounds of theinvention are within the scope of the invention. Additionally, unlessotherwise stated, structures depicted herein are also meant to includecompounds that differ only in the presence of one to five isotopicallyenriched atoms. For example, compounds having the present structuresexcept for the replacement of hydrogen by deuterium or tritium, or thereplacement of a carbon by a ¹³C— or ¹⁴C-enriched carbon are within thescope of this invention. Such compounds are useful, for example, asanalytical tools or probes in biological assays.

In a first aspect, the present invention features a compound having theformula:

or a pharmaceutically acceptable salt or prodrug thereof, wherein:

-   R¹ is halogen, —CN, —NO₂, —NR₂, —OR, —SR, or an optionally    substituted C₁₋₄ aliphatic or C₃₋₆ cycloaliphatic;-   R² is hydrogen or C₁₋₃ aliphatic;-   each R³ is, independently, hydrogen, halogen, —NR₂, —OR, —SR, or an    optionally substituted C₁₋₄ aliphatic group or C₃₋₆ cycloaliphatic    group, or two R³, taken together with the intervening carbon atom,    form an optionally substituted 3-6 membered cycloaliphatic or    heterocyclyl ring having 1-2 heteroatoms selected from nitrogen,    oxygen, or sulfur;-   X¹ is CR⁴ or N, wherein R⁴ is hydrogen, halogen, —C(O)R, —C(S)R,    —C(O)N(R)₂, —C(S)N(R)₂, —CN, —NO₂, or an optionally substituted C₁₋₄    aliphatic group;-   A¹ is a phenyl ring or a 5-6 membered heteroaryl ring having 1-3    heteroatoms independently selected from nitrogen, oxygen, or sulfur,    wherein said phenyl or heteroaryl ring is optionally substituted    with 1-5 independent occurrences of TR⁵, wherein two TR⁵ groups are    optionally taken together to form methylenedioxy or ethylenedioxy;-   T is a bond or a C₁-C₆ alkylidene chain, wherein up to two methylene    units of T are optionally and independently replaced by —NR′—, —S—,    —O—, —C(S)—, —C(O)O—, —OC(O)—, —C(O)—, —C(O)C(O)—, —C(O)NR′—,    —NR′C(O)—, —NR′C(O)O—, —S(O)₂NR′—, —NR′S(O)₂—, —C(O)NR′NR′—,    —NR′C(O)NR′-, —OC(O)NR′, —NR′NR′—, —NR′S(O)₂NR′-, —S(O)—, —S(O)₂—,    —P(O)—, —P(O)₂—, —P(O)R′;-   each R⁵ is independently R^(a), halogen, —NO₂, or —CN;-   each occurrence of R^(a) is, independently, hydrogen, an optionally    substituted moiety selected from a C₁₋₄ aliphatic group, a C₃₋₆    cycloaliphatic group, a C₁₋₄ haloaliphatic group, a 6-10 membered    mono- or bicyclic aryl ring, a 3-10 membered mono- or bicyclic    cycloaliphatic ring, or a 5-14 membered mono- or bicyclic heteroaryl    or heterocyclyl ring having 1-5 heteroatoms independently selected    from nitrogen, oxygen, or sulfur, or wherein R^(a), R′, and an    intervening nitrogen atom together form an optionally substituted    5-6 membered heterocyclic or heteroaryl ring optionally having 1-2    additional heteroatoms selected from nitrogen, oxygen, or sulfur;-   each optionally substituted group or ring is, independently,    optionally substituted with 1-5 substituents independently selected    from C₁₋₄ aliphatic, C₃₋₆ cycloaliphatic, C₁₋₄ haloaliphatic,    halogen, —OR″, —OC(O)N(R″)₂, —OC(O)R″, —OC(O)OR″, —NO₂, —N(R″)₂,    —NR″C(O)R″, —NR″C(S)R″, —NR″C(O)N(R″)₂, —NR″C(S)N(R″)₂, —NR″C(O)OR″,    —NR″NR″C(O)R″, —NR″NR″C(O)N(R″)₂, —NR″NR″C(O)OR″, —NR″S(O)₂N(R″)₂,    —NR″S(O)₂R″, —N(OR″)R″, —CN, C(O)OR″, —C(O)R″, —C(S)R″, —C(O)N(R″)₂,    —C(S)N(R″)₂, —OC(O)N(R″)₂, —OC(O)R″, —C(O)N(OR″)R″, —C(═NOR″)R″,    —C(═NH)—N(R″)₂, —SR″, —SC(O)R″, —SC(S)R″, —S(O)R″, —S(O)₂R″,    —S(O)₂OR″, or —S(O)₂N(R″)₂;-   each occurrence of R is, independently, hydrogen, a C₁₋₄ aliphatic    group, a C₃₋₆ cycloaliphatic group, —(CH₂)₁₋₂Ph, —CH═CHPh, or a C₁₋₄    haloaliphatic group, or two R groups and an intervening nitrogen    atom together form an optionally substituted 5-6 membered    heterocyclic or heteroaryl ring optionally having 1-2 additional    heteroatoms selected from nitrogen, oxygen, or sulfur;-   each occurrence of R′ is, independently, hydrogen, a C₁₋₄ aliphatic    group, a C₃₋₆ cycloaliphatic group, —(CH₂)₁₋₂Ph, —CH═CHPh, or a C₁₋₄    haloaliphatic group, or two R′ groups and an intervening nitrogen    atom together form an optionally substituted 5-6 membered    heterocyclic or heteroaryl ring optionally having 1-2 additional    heteroatoms selected from nitrogen, oxygen, or sulfur; and-   each occurrence of R″ is, independently, hydrogen, a C₁₋₄ aliphatic    group, a C₃₋₆ cycloaliphatic group, —(CH₂)₁₋₂Ph, —CH═CHPh, or a C₁₋₄    haloaliphatic group, or two R″ groups and an intervening nitrogen    atom together form an optionally substituted 5-6 membered    heterocyclic or heteroaryl ring optionally having 1-2 additional    heteroatoms selected from nitrogen, oxygen, or sulfur.

In one embodiment, R² is hydrogen.

In another embodiment, A¹ is a phenyl ring or a 5-6 membered heteroarylring having 1-2 heteroatoms independently selected from nitrogen,oxygen, or sulfur, wherein said phenyl or heteroaryl ring is optionallysubstituted with 1-3 independent occurrences of TR⁵, wherein two TR⁵groups are optionally taken together to form methylenedioxy orethylenedioxy.

Examples of compounds of formula I wherein A¹ is a phenyl ring includecompounds having the formula:

-   R¹ is C₁₋₄ aliphatic, halogen, —NR₂, —OR, or —SR;-   each T is, independently, a bond or a C₁-C₆ alkylidene chain,    wherein up to two methylene units of T are optionally and    independently replaced by —NR′—, —S—, —O—, —C(O)NR′—, —NR′C(O)—,    —NR′C(O)O—, —S(O)₂NR′—, —NR′S(O)₂—, —NR′C(O)NR′—, —OC(O)NR′—, or    —NR′S(O)₂NR′—, or two TR⁵ groups are optionally taken together to    form methylenedioxy or ethylenedioxy;-   each R⁵ is, independently, R^(a) or halogen; and-   n is 1 to 3.

In one embodiment for any compound of formula I or I-A, one of −TR⁵ isat the 3-position. In some examples, −TR⁵ at the 3-position is chloro,fluoro, —OH, optionally substituted C₁₋₄ alkoxy, —NHS(O)₂R^(a),—S(O)₂NRR^(a), or two TR⁵ groups at the 3 and 4 positions together aremethylenedioxy.

In another embodiment for any compound of formula I or I-A, R¹ ishalogen, such as, for example, chloro or fluoro; —NR₂, such as, forexample, NH₂, NHCH₃, N(CH₃)₂, NHCH₂CH₃, or NHCH₂Ph; —OR, such as, forexample, OCH₃ or OCH₂CH₃; or an optionally substituted C₁₋₄ aliphaticgroup, such as, for example, CH₃ or CH₂CH₃.

In yet another embodiment for any compound of formula I or I-A, each R³is, independently, hydrogen, halogen, —NR′″₂, —OR′″, —SR′″, or a C₁₋₄aliphatic group, wherein each R′″ is, independently, hydrogen or a C₁₋₄aliphatic group. Examples include those in which R³ is hydrogen, chloro,fluoro, or —OH.

For some compounds of formula I or I-A, X¹ is CR⁴, wherein R⁴ ishydrogen or a C₁₋₄ aliphatic group. For other compounds of formula I orI-A, X¹ is N.

In another aspect, the invention features a compound selected from thelist of compounds in Table 1. TABLE 1 Compound No. Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

Formulation, Uses, and Administration

The compounds and compositions described herein are generally useful forthe inhibition of protein kinase activity of one or more enzymes. In oneparticular embodiment, the compounds and compositions of the inventionare inhibitors of ROCK and thus the compounds and compositions areparticularly useful for treating or lessening the severity of disease ordisease symptoms associated with ROCK.

The activity of a compound utilized in this invention as an inhibitor ofROCK may be assayed in vitro, in vivo or in a cell line. In vitro assaysinclude assays that determine inhibition of either the phosphorylationactivity or ATPase activity of activated ROCK. Alternate in vitro assaysquantitate the ability of the inhibitor to bind to ROCK. Inhibitorbinding may be measured by radiolabeling the inhibitor prior to binding,isolating the inhibitor/ROCK complex and determining the amount ofradiolabel bound. Alternatively, inhibitor binding may be determined byrunning a competition experiment where new inhibitors are incubated withROCK bound to known radioligands. Detailed conditions for assaying acompound utilized in this invention as an inhibitor of ROCK kinase areset forth in the Examples below.

In one embodiment, a compound of formula I or formula IA binds to ROCKwith a K_(i) of less than or equal to 0.1 micromolar as measured invitro and inhibits at least one cytochrome P450 isozyme (such as, forexample, CYP1A2, CYP2C19, CYP2C9, CYP2D6, or CYP3A4) with an IC₅₀ ofgreater than or equal to 5 micromolar as measured in vitro. In otherembodiments, a compound of formula I or formula IA binds to ROCK with aK_(i) of less than or equal to 0.1 micromolar and inhibits 3, 4, or allof the cytochrome P450 isozymes selected from the group consisting ofCYP1A2, CYP2Cl₉, CYP2C9, CYP2D6, and CYP3A4 with an IC₅₀ of greater thanor equal to 10 micromolar.

According to another aspect, the invention features a compositioncomprising a compound of this invention or a pharmaceutically acceptablesalt or prodrug thereof, and a pharmaceutically acceptable carrier,adjuvant, or vehicle. The amount of compound in the compositions of thisinvention is such that is effective to detectably inhibit a proteinkinase, particularly ROCK, in a biological sample or in a patient.Preferably the composition of this invention is formulated foradministration to a patient in need of such composition. Mostpreferably, the composition of this invention is formulated for oraladministration to a patient.

The term “patient,” as used herein, means an animal, preferably amammal, and most preferably a human.

As described above, the pharmaceutically acceptable compositions of thepresent invention additionally comprise a pharmaceutically acceptablecarrier, adjuvant, or vehicle, which, as used herein, includes any andall solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. In Remington: TheScience and Practice of Pharmacy, 21 st edition, 2005, ed. D. B. Troy,Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia ofpharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,1988-1999, Marcel Dekker, New York, the contents of each of which isincorporated by reference herein, are disclosed various carriers used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to a non-toxic carrier, adjuvant, or vehicle that does notdestroy the pharmacological activity of the compound with which it isformulated. Pharmaceutically acceptable carriers, adjuvants or vehiclesthat may be used in the compositions of this invention include, but arenot limited to, ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

The term “detectably inhibit,” as used herein, means a measurable changein ROCK activity between a sample comprising a compound or compositionof the invention and a ROCK kinase and an equivalent sample comprisingROCK kinase in the absence of the compound or composition.

A “pharmaceutically acceptable derivative” means any non-toxic salt,ester, salt of an ester or other derivative of a compound of thisinvention that, upon administration to a recipient, is capable ofproviding, either directly or indirectly, a compound of this inventionor an inhibitorily active metabolite or residue thereof.

As used herein, the term “inhibitorily active metabolite or residuethereof” means that a metabolite or residue thereof is also an inhibitorof ROCK.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptanoate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate,palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, salicylate, succinate, sulfate, tartrate,thiocyanate, tosylate and undecanoate. Other acids, such as oxalic,while not in themselves pharmaceutically acceptable, may be employed inthe preparation of salts useful as intermediates in obtaining thecompounds of the invention and their pharmaceutically acceptable acidaddition salts.

Salts derived from appropriate bases include alkali metal (e.g., sodiumand potassium), alkaline earth metal (e.g., magnesium), ammonium andN⁺(C₁₋₄ alkyl)₄ salts. This invention also envisions the quaternizationof any basic nitrogen-containing groups of the compounds disclosedherein. Water or oil-soluble or dispersible products may be obtained bysuch quaternization.

The compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term“parenteral,” as used herein, includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques. Preferably, the compositions are administeredorally, intraperitoneally or intravenously. Sterile injectable forms ofthe compositions of this invention may be aqueous or oleaginoussuspension. These suspensions may be formulated according to techniquesknown in the art using suitable dispersing or wetting agents andsuspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

The pharmaceutically acceptable compositions of this invention may beorally administered in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient that is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

The pharmaceutically acceptable compositions of this invention may alsobe administered topically, especially when the target of treatmentincludes areas or organs readily accessible by topical application,including diseases of the eye, the skin, or the lower intestinal tract.Suitable topical formulations are readily prepared for each of theseareas or organs.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutically acceptable compositionsmay be formulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutically acceptable compositions canbe formulated in a suitable lotion or cream containing the activecomponents suspended or dissolved in one or more pharmaceuticallyacceptable carriers. Suitable carriers include, but are not limited to,mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax,cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutically acceptable compositions may beformulated as micronized suspensions in isotonic, pH adjusted sterilesaline, or, preferably, as solutions in isotonic, pH adjusted sterilesaline, either with or without a preservative such as benzylalkoniumchloride. Alternatively, for ophthalmic uses, the pharmaceuticallyacceptable compositions may be formulated in an ointment such aspetrolatum.

The pharmaceutically acceptable compositions of this invention may alsobe administered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

Preferably, the pharmaceutically acceptable compositions of thisinvention are formulated for oral administration.

The amount of the compounds of the present invention that may becombined with the carrier materials to produce a composition in a singledosage form will vary depending upon the host treated, the particularmode of administration. Preferably, the compositions should beformulated so that a dosage of between 0.01-100 mg/kg body weight/day ofthe inhibitor can be administered to a patient receiving thesecompositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

According to another aspect, the invention features a method ofinhibiting protein kinase activity, such as, for example, ROCK kinaseactivity, in a biological sample comprising the step of contacting thebiological sample with a compound of this invention, or a compositioncomprising the compound.

The term “biological sample,” as used herein, means a sample outside ananimal and includes, without limitation, cell cultures or extractsthereof, biopsied material obtained from an animal or extracts thereof,and blood, saliva, urine, feces, semen, tears, or other body fluids orextracts thereof. Inhibition of kinase activity, particularly ROCKkinase activity, in a biological sample is useful for a variety ofpurposes known to one of skill in the art. Examples of such purposesinclude, but are not limited to, biological specimen storage andbiological assays.

In another aspect, the invention features a method of inhibiting proteinkinase activity, such as, for example, ROCK kinase activity, in apatient comprising the step of administering to the patient a compoundof the present invention, or a composition comprising the compound.

According to another embodiment, the invention provides a method fortreating or lessening the severity of an ROCK-mediated disease orcondition in a patient comprising the step of administering to thepatient a composition according to the present invention.

The term “ROCK-mediated disease” or “condition,” as used herein, meansany disease or other deleterious condition in which ROCK is known toplay a role. ROCK participates in a variety of important physiologicalfunctions in the vasculature, including smooth muscle contraction; cellproliferation, such as, for example, vascular smooth muscle cellproliferation; and cell adhesion and migration (see Hu & Lee, ExpertOpin. Ther. Targets 9(4):715-36, 2005; Shimokawa & Takeshita,Arterioscler. Thromb. Vasc. Biol. 25(9):1767-75, 2005). ROCK isparticipates in inflammatory responses due to leukocyte migration, suchas, for example, autoimmune disease and allergic reactions (seeWettschureck et al., J. Mol. Med. 80:629-38, 2002). Abnormal activationof the Rho/ROCK pathway has been observed in various disorders of thecentral nervous system (see Mueller et al, Nature Rev. 4:387-98, 2005).In addition, ROCK has been implicated in tumor cell migration andinvasion (Riento & Ridley, Nature Rev. 4:446-56, 2004) and inosteoporosis (Ohnaka et al., Biochem. Biophys. Res. Commun.287(2):337-4, 2001).

Accordingly, another embodiment of the present invention relates totreating or lessening the severity of a disease or disorder in whichROCK is known to play a role. Specifically, the present inventionrelates to a method of treating or lessening the severity of acardiovascular disease or disorder, such as, for example, cerebralvasospasm, hypertension, atherosclerosis, angina, myocardial infarction,ischemic/reperfusion injury, stroke, bronchial asthma; glaucoma,pre-term labor, erectile dysfunction, or renal disease, such as, forexample, chronic renal failure, chronic nephritis, diabetic nephropathy,and IgA nephropathia; a neurological disease or disorder, such as forexample, spinal-cord injury, Alzheimer's disease, multiple sclerosis, orneuropathic pain; and proliferative disorders, such as, for example,retinopathy, fibrosis, or invasive/metastatic cancers. Such cancersinclude adenocarcinoma, adrenocortical cancer; bladder cancer; bonecancer; brain cancer; breast cancer; cancer of the buccal cavity;cervical cancer; colon cancer; colorectal cancer; endometrial or uterinecarcinoma; epidermoid carcinoma; esophogeal cancer; eye cancer;follicular carcinoma; gallbladder cancer; gastrointestinal cancer;cancer of the genitourinary tract; glioblastoma; hairy cell carcinoma;head and neck cancer; hepatic carcinoma; hepatocellular cancer;Hodgkin's disease; keratoacanthoma; kidney cancer; large cell carcinoma;cancer of the large intestine; laryngeal cancer; liver cancer; lungcancer, such as, for example, adenocarcinoma of the lung, small-celllung cancer, squamous carcinoma of the lung, non-small cell lung cancer;melanoma; myeloproliferative disorders, neuroblastoma; ovarian cancer;papillary carcinoma; pancreatic cancer; cancer of the peritoneum;prostate cancer; rectal cancer; salivary gland carcinoma; sarcoma;squamous cell cancer; small cell carcinoma; cancer of the smallintestine; stomach cancer; testicular cancer; thyroid cancer; and vulvalcancer. In particular embodiments, the treated cancer is melanoma,breast cancer, colon cancer, or pancreatic cancer.

The treatment method that includes administering an ROCK inhibitor ofthe invention can further include administering to the patient anadditional therapeutic agent (combination therapy) selected from: achemotherapeutic or anti-proliferative agent, an anti-inflammatoryagent, an immunomodulatory or immunosuppressive agent, a neurotrophicfactor, an agent for treating cardiovascular disease, an agent fortreating destructive bone disorders, an agent for treating renaldisease, or an agent for treating blood disorders, wherein theadditional therapeutic agent is appropriate for the disease beingtreated and the additional therapeutic agent is administered togetherwith a compound or composition of the invention as a single dosage formor separately from the compound or composition as part of a multipledosage form. The additional therapeutic agent may be administered at thesame time as a compound of the invention or at a different time. In thelatter case, administration in normally within 5 hours or each other butmay be staggered by, for example, 6 hours, 12 hours, 1 day, 2 days, 3days, 1 week, 2 weeks, 3 weeks, 1 month, or 2 months. Non-limitingexamples of chemotherapeutic agents or other anti-proliferative agentsthat may be combined with a compound of this invention includeadriamycin, gemcitabine, cyclophosphamide, dexamethasone, etoposide,fluorouracil, Gleevec™, interferons, platinum derivatives, such ascarboplatin, topotecan, taxol, vinblastine, and vincristine.Non-limiting examples of immunomodulatory agents that may be combinedwith a compound of this invention include alpha-, beta-, andgamma-interferons, pegylated derivatized interferon-α compounds,ribaviron, and thymosin. Non-limiting examples of immunosuppressiveagents that may be combined with a compound of this invention includecyclosporin, tacrolimus, rapamycin, mycophenolate mofetil, interferons,corticosteroids, cyclophosphamide, azathioprine, and sulfasalazine.Non-limiting examples of neurotrophic factors that may be combined witha compound of this invention include acetylcholinesterase inhibitors,MAO inhibitors, interferons, anti-convulsants, ion channel blockers,riluzole, and anti-Parkinsonian agents. Non-limiting examples of agentsfor treating cardiovascular disease that may be combined with a compoundof this invention include beta-blockers, ACE inhibitors, diuretics,nitrates, calcium channel blockers, and statins. Non-limiting examplesof agents for treating blood disorders that may be combined with acompound of this invention include corticosteroids, anti-leukemicagents, and growth factors.

The amount of compound of the invention or the amount of compound andadditional therapeutic agent (in those compositions which comprise anadditional therapeutic agent as described above)) that may be combinedwith the carrier materials to produce a single dosage form will varydepending upon the host treated and the particular mode ofadministration. Preferably, the compositions of this invention should beformulated so that a dosage of between 0.01-100 mg/kg body weight/day ofa compound of formula I can be administered.

In those compositions that include an additional therapeutic agent, thatadditional therapeutic agent and the compound of this invention may actsynergistically. Therefore, the amount of additional therapeutic agentin such compositions will be less than that required in a monotherapyutilizing only that therapeutic agent. In such compositions a dosage ofbetween 0.01-100 μg/kg body weight/day of the additional therapeuticagent can be administered.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

The compounds of this invention, or pharmaceutical compositions thereof,may also be incorporated into compositions for coating an implantablemedical device, such as prostheses, artificial valves, vascular grafts,stents and catheters. Vascular stents, for example, have been used toovercome restenosis (re-narrowing of the vessel wall after injury).However, patients using stents or other implantable devices risk clotformation or platelet activation. These unwanted effects may beprevented or mitigated by pre-coating the device with a pharmaceuticallyacceptable composition comprising a kinase inhibitor. Suitable coatingsand the general preparation of coated implantable devices are describedin U.S. Pat. Nos. 6,099,562; 5,886,026; and 5,304,121. The coatings aretypically biocompatible polymeric materials such as a hydrogel polymer,polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylacticacid, ethylene vinyl acetate, and mixtures thereof. The coatings mayoptionally be further covered by a suitable topcoat of fluorosilicone,polysaccarides, polyethylene glycol, phospholipids or combinationsthereof to impart controlled release characteristics in the composition.Implantable devices coated with a compound of this invention are anotherembodiment of the present invention.

Preparation of the Compounds of the Invention

The compounds of the present invention may be prepared according tomethods known to one or ordinary skill in the art and by those describedin U.S. patent application No. 20040122016, the entirety of which ishereby incorporated by reference. Although certain exemplary embodimentsare depicted and described herein, it will be appreciated that compoundsof the invention can be prepared as generally described herein usingappropriate starting materials that are commercially available orobtained by methods generally available to one of ordinary skill in theart. In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

The following definitions describe terms and abbreviations used herein:

-   ATP adenosine triphosphate-   Boc tert-butoxycarbamate-   BtS(O)₂Me N-(1-methanesulfonyl)benzotriazole-   DMF dimethylformamide-   ESMS electrospray mass spectrometry-   HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-   HPLC high performance liquid chromatography-   LCMS liquid chromatography-mass spectrometry-   Me methyl-   MeOH methanol-   NADH nicotinamide adenine dinucleotide hydride-   Ph phenyl-   tBu tertiary butyl-   TFA trifluoacetic acid-   THF tetrahydrofuran

Scheme 1 shows a general synthetic route useful for preparing compoundsof formula I of this invention.

Accordingly, a compound of formula II, where R¹, R², and X¹ are asdescribed herein, is reacted to a compound of formula III, where R³ isas described herein and L is a leaving group. For example, L can be ahalogen or an activated hydroxyl. Methods for activating hydroxyls arewell known to those skilled in the art and include the use ofconventional condensation reagents including, for example,1-benzotriazol-1-yloxy-bis(pyrrolidino)uronium hexafluorophosphate(BBC), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU),O-(7-azabenzotriazol-1-yl)-1,1,3,3-bis(tetramethylene)uroniumhexafluorophosphate (HATU),O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), 1,3-diisopropylcarbodiimide (DIC),1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride,(EDC)O-(7-azabenzotriazol-1-yl)-tris(dimethylamino)phosphoniumhexafluorophosphate (AOP),1-benzotriazolyoxytris(dimethylamino)phosphonium hexafluorophosphate(BOP), 7-azobenzotriazolyoxytris(pyrrolidino)phosphoniumhexafluorophosphate (PyABOP), and1-benzotriazolyoxytris(pyrrolidino)phosphonium hexafluorophosphate(PyBOP).

Synthetic procedures analogous to those used in the preparations ofcompounds of formulae IV, V, and VI, where R¹ is hydrogen, n is 1 to 3,and TR⁵ is as defined herein, as well as their subsequent use to formcompounds of formulae I and II, are found in U.S. patent applicationPublication No. 20040122016, which procedures are hereby incorporated byreference.

In another general example, thiazole-containing compounds of formula IX,where R¹ and A¹ are as defined herein, can be prepared as shown inScheme 2, where a bromoacetyl compound of formula VII is reacted with athiourea of formula VIII to produce a thiazole of formula IX.

In yet another general example, thiophene-containing compounds offormula XIII, where R¹, n, and TR⁵ are as defined herein, can beprepared as shown in Scheme 3. Accordingly, a boronic acid or boronicacid derivative of formula X is reacted with a thiophene of formula XI,where P is a suitable nitrogen protecting group (e.g., Boc), in apalladium catalyzed coupling reaction to form a compound of formula XII.The compound of formula XII is then coupled to an optionally substitutedphenylacetic acid of formula XI to form a compound of formula XIII. Inthis example, the coupling is affected in the presence ofN-(1-methanesulfonyl)benzotriazole (BtS(O)₂Me) by the application ofmicrowave irradiation and heat.

EXAMPLES

The following examples provide detailed methods for preparing exemplarycompounds of the present invention. It will be appreciated that othercompounds of the present invention are prepared in accordance with theteachings provided herein and with methods known to one or ordinaryskill in the art.

Example 1 Preparation of Compound 18

Compound 18 was prepared as outlined in Scheme 4. Accordingly, potassiumt-butoxide (6.1 g) was added slowly to a stirred solution oftert-butylnitrite (5.5 mL) and 2-fluoro-4-methylpyridine (compound 1001,4.0 g) in 75 mL of THF at 0° C. After the addition was complete, thereaction mixture was stirred at room temperature for 2 hours. Thereaction mixture was concentrated in vacuo and the crude productextracted with ethyl acetate (4×50 mL). The resulting solid was washedwith hexanes to yield (2-fluoropyridin-4-yl)methan-N-hydroxyimine(compound 1002; 4.0 g; ESMS: M+H⁺=141). Thionyl chloride (2.6 mL) wasadded slowly to a stirred solution of compound 1002 (4.0 g in 40 mL ofTHF) at 0° C. After stirring for 1 hour at room temperature, thevolatiles were removed in vacuo. The residue was taken up in ethylacetate (50 mL) and the organics were washed with water and brine. Theorganics were concentrated in vacuo to give2-fluoropyridine-4-carbonitrile (compound 1003) as a white solid (3.0 g;ESMS: M+H⁺=123). Compound 1003 (1.0 g) and 4-methoxybenzylamine werecombined and heated at 130° C. for 1 hour. The crude product waspartitioned between water and ethyl acetate (50 mL). The aqueous layerwas extracted with ethyl acetate and the combined organics dried overNa₂SO₄, concentrated, and recrystallized from ethyl acetate/hexanes toprovide 2-(4-methoxybenzylamino)pyridine-4-carbonitrile (compound 1004;1.7 g; ESMS: M+H⁺=238) as pale yellow solid. Compound 1004 (1.6 g) wasdissolved in 50 mL of anhydrous ether and 9 mL of 3M methyl magnesiumbromide in ether was added. The reaction mixture was stirred at roomtemperature for 16 hours and poured over ice (c. 50 g), which wassubsequently acidified with 6M HCl, followed by basicification with 1Mammonium hydroxide. The resulting mixture was extracted with ethylacetate (3×50 mL). The combined organics were concentrated in vacuo andpurified by silica gel chromatography to provide1-(2-(4-methoxybenzylamino)pyridin-4-yl)ethanone (Compound 1005; 1.2 g;ESMS: M+H⁺=257) as a yellow solid.

Separately, to 2-(3-methoxyphenyl)acetic acid (compound 1007, 1 g) in 25mL of dichloromethane containing 0.1 mL of DMF was added 2.45 mL of 2Mphosgene/CH₂Cl₂. The reaction mixture was stirred for 3 hours at roomtemperature and the volatiles were removed in vacuo. The residue wastreated with 25 mL of THF and 1.82 g of thiourea, followed by heating at80° C. for 3 hours. The mixture was cooled, poured into 20 mL of water,which was extracted with ethyl acetate (3×50 mL). The combined organicswere concentrated and the residue recrystallized (ethyl acetate/hexanes)to provide 1-(2-(3-methoxyphenyl)acetyl)thiourea (compound 1008, 476mg).

Compound 1005 (50 mg) was dissolved in 0.9 mL of acetic acid containing0.2 mL of 48% HBr. Bromine (10 L) was added and the reaction mixture washeated at 70° C. for 2 hours to produce intermediate compound 1006(bromination of the acetyl group was accompanied by loss of the4-methoxybenzyl protecting group), which was not isolated. Compound 1008(44 mg in 7 mL of ethanol) was added and the reaction mixture heated at70° C. for 1 hour. The reaction mixture was cooled, poured into 10 mL ofwater and the solution made basic with concentrated ammonium hydroxide.The mixture was concentrated in vacuo and the residue purified byreversed-phase HPLC (10-90% acetonitrile/water gradient containing 0.1%TFA) to provideN-(4-(2-aminopyridin-4-yl)thiazol-2-yl)-2-(3-methoxyphenyl)acetamide(compound 18, 36 mg; ESMS: M+H⁺=341).

Example 2 Preparation of Compound 62

As shown in Scheme 5, a mixture of 4-bromothiophene-2-carboxylic acid(53 g, 255 mmol), diphenylphosphoryl azide (70 mL, 323 mmol), andtriethylamine (45 mmol) in tertiary butanol (675 mL) was heated at 100°C. for 5 hours. After cooling to room temperature, the volatiles wereremoved in vacuo to give a brown gum, which was dissolved in ethylacetate (500 mL). The organics were washed with saturated NaHCO₃ andwater, followed by drying over Na₂SO₃ and concentration in vacuo. Theresidue was dissolved in a minimum amount of CH₂Cl₂ and purified bychromatography on silica gel (10-15% EtOAc/hexanes) to producetert-butyl 4-bromothiophen-2-ylcarbamate as a white solid (compound1010, 47 g, ESMS: M+H⁺=2781). Compound 1010 (96.7 mg, 0.35 mmol) wastreated with2-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine(compound 1011, obtained from 2-chloro-4-bromopyridine by lithiation andsubsequent reaction with2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaborolane), a catalytic amount oftetrakis(triphenylphosphine)palladium(0), and potassium carbonate (2.06mmol, 284 mg) in dioxane/water (2.4 mL/0.8 mL) to produce tert-butyl4-(2-chloropyridin-4-yl)thiazol-2-ylcarbamate (compound 1012, ESMS:M+H⁺=311.5). The reaction mixture was dissolved in ethyl acetate, (10mL) and washed with NaHCO₃ and H₂O. After drying, the solvent wasremoved in vacuo and the mixture carried forward without furtherpurification. Compound 1012 was treated with 4M HCl/dioxane (3 mL) atroom temperature for 2 hours. The resulting precipitate was filtered toproduce 4-(2-chloropyridin-4-yl)thiazol-2-amine HCl salt (compound 1013,ESMS: M+H⁺=211.5, 85% yield). Compound 1013 (30 mg, 0.14 mmol) wascombined with 2-(3-methoxyphenyl)acetic acid (28 mg, 0.17 mmol),N-(1-methanesulfonyl)benzotriazole (55 mg, 0.28 mmol) and triethylamine(41 μL, 0.30 mmol) in 2 mL THF and the mixture irradiated with microwaveradiation for 10 minutes at 160° C. After removal of the solvent, thereaction mixture was taken into ethyl acetate, washed with 2N NaOH, andwater. After removal of the solvent, the reaction mixture was purifiedby reversed-phase HPLC to producedN-(4-(2-chloropyridin-4-yl)thiazol-2-yl)-2-(3-methoxyphenyl)acetamide(compound 62, ESMS: M+H⁺=359, 33% yield).

Example 3 Preparation of Compound 64

As shown in Scheme 6, 2.5 M n-BuLi (7.2 mL, 18 mmol) was added dropwiseto a stirred solution of tert-butyl 4-bromothiophen-2-ylcarbamate(compound 1009, 1.0 g, 3.6 mmol) in 3 mL of THF and 11 mL of toluene at−78° C. The mixture was stirred at −78° C. for 1 hour and triisopropylborate (2.0 mL, 9 mmol) was added. Stirring was continued at −78° C. for30 minutes, followed by stirring at room temperature for 30 minutes.Ethyl acetate (50 mL) and 2M HCl (15 mL) were added to the reactionmixture and stirring was continued for 25 minutes. The organic layer wasseparated and the aqueous layer was extracted with ethyl acetate (2×25mL). The combined organics were dried and concentrated in vacuo toprovide 5-(tert-butoxycarbamyl)thiphen-3-yl-3-boronic acid as an oil(compound 1015, 0.8 g, ESMS: M+H⁺=242), which was used in the next stepas is without any subsequent purification. Compound 1015 was dissolvedin 4M HCl/dioxane (10 mL) and methanol (2 mL) and the reaction mixturestirred at room temperature for 2 hours. The volatiles were removed invacuo to provide 5-aminothiophen-3-yl-3-boronic acid (compound 1016,0.58 g, ESMS: M+H⁺=143) as a brown gum. This intermediate was used as iswithout any subsequent purification. Compound 1016 (50 mg, 0.34 mmol)and triethylamine (50 μL) in 3 mL of DMF at room temperature was treatedwith and 2-(3-methoxyphenyl)acetyl chloride (0.408 mmol). The reactionmixture was stirred at room temperature overnight. The reaction mixturewas dissolved in ethyl acetate (2×5 mL) and extracted with 1N NaOH. Thebasic extracts were acidified with 6N HCl, extracted with EtOAc (2×5mL), and washed with water and brine. After drying over Na₂SO₄, thesolvent was evaporated in vacuo to provide5-(2-(3-methoxyphenyl)acetamido)thiophen-3-yl-3-boronic acid (compound1017, ESMS: M+H⁺=292). Compound 1017 (from previous step) and tert-butyl4-chloropyridin-2-ylcarbamate (compound 1018, 0.34 mmol) were dissolvedin dioxane/water (3 mL/1 mL) under an atmosphere of nitrogen inmicrowave tube. A catalytic around of trisdibenzylideneacetonepalladium(0) was added, followed by the addition of three equivalents ofpotassium fluoride (and tri(tert-butyl)phosphine (0.045 mole %). Thereaction mixture was heated under microwave irradiation at 180° C. for15 min. After cooling, the organic layer was concentrated and the crudeproduct dissolved in DMF and water, followed by reversed-phase HPLCpurification to provideN-(4-(2-aminopyridin-4-yl)thiophen-2-yl)-2-(3-methoxyphenyl)acetamide(compound 67, 10 mg, 8.6% yield from 1016), ESMS: M+H⁺=340).

Spectral data for selected compounds of the invention are presented inTable 1. TABLE 1 Cmpd ¹H-NMR(500 MHz) ESMS No. NMR peaks given as δvalues (M+1) 1 (DMSO-d₆): 12.57(s, 1H), 8.45(d, 1H), 8.11(s, 1H), 3607.94(s, 1H), 7.85(dd, 1H), 7.25(t, 1H), 6.83-6.93(m, 3H), 3.77(s, 2H),3.75(s, 3H) 2 (methanol-d₄): 8.34(d, 1H), 7.95(s, 1H), 7.82-7.84(m, 2H),348 7.36(m, 2H), 7.06(m, 2H), 3.79(s, 2H) 3 (DMSO-d₆): 12.53(s, 1H),8.20(d, 1H), 7.95(s, 1H), 356 7.45(dd, 1H), 7.24(m, 2H), 6.82-6.84(m,3H), 3.90(s, 3H), 3.76(s, 2H), 3.74(s, 3H) 4 (CDCl₃): 12.50(brs, 1H),8.23(d, 1H), 8.11(s, 1H), 344 7.14-7.30(m, 5H), 6.98(t, 2H), 3.95(s,3H), 3.79(s, 2H) 5 (methanol-d₄): 8.17(d, 1H), 7.93(s, 1H), 7.66(dd,1H), 370 7.58(s, 1H), 7.24(t, 1H), 6.92(m, 2H), 6.83(dd, 1H), 4.45(q,2H), 3.79(s, 3H), 3.76(s, 2H), 1.47(t, 3H) 6 (DMSO-d₆): 12.52(s, 1H),8.18(d, 1H), 7.94(s, 1H), 418 7.43(dd, 1H), 7.23(s, 1H), 7.08(s, 1H),7.03(s, 1H), 6.07(s, 2H), 4.32(q, 2H), 1.33(t, 3H) 7 (methanol-d₄):8.20(d, 1H), 8.03(s, 1H), 7.75(dd, 1H), 358 7.69(s, 1H), 7.37(m, 2H),7.06(m, 2H), 4.49(q, 2H), 3.80(s, 2H), 1.50(t, 3H) 8 (methanol-d₄):8.21(d, 1H), 8.05(s, 1H), 7.77(d, 1H), 374 7.72(s, 1H), 7.41(m, 2H),7.30(m, 2H), 4.51(q, 2H), 4.01(s, 2H), 1.51(t, 3H) 9 (DMSO-d₆): 12.51(s,1H), 8.10(d, 1H), 7.83(s, 1H), 369 7.23(d, 1H), 7.08(s, 1H), 7.03(d,1H), 6.82-6.85(m, 3H), 3.79(s, 2H), 3.75(s, 3H), 3.06(s, 6H) 10(DMSO-d₆): 12.7(brs, 1H), 8.46(d, 1H), 8.13(s, 1H), 364 7.91(s, 1H),7.86(d, 1H), 7.46(m, 2H), 7.33(m, 2H), 4.01(s, 2H) 11 (methanol-d₄):8.03(d, 1H), 7.60(s, 1H), 7.40(m, 2H), 373 7.30(m, 2H), 7.46(m, 2H),7.22(s, 1H), 7.07(s, 1H), 3.99(s, 1H), 3.11(s, 6H) 12 (methanol-d₄):8.04(s, 1H), 7.88(d, 1H), 7.67(s, 1H), 417 7.43(d, 1H), 6.91(s, 1H),6.90(s, 1H), 6.00(s, 2H), 3.90(s, 2H), 3.33(s, 6H) 13 (DMSO-d₆):12.58(s, 1H), 8.28(d, 1H), 8.10(s, 1H), 344 7.80(d, 1H), 7.56(s, 1H),7.23(t, 1H), 6.83-6.93(m, 3H), 3.77(s, 2H), 3.75(s, 3H) 14 (DMSO-d₆):12.45(s, 1H), 7.97(d, 1H), 7.68(s, 1H), 431 6.84-7.34(m, 12H), 4.52(d,2H), 3.75(s, 2H), 3.73(s, 3H) 15 (DMSO-d₆): 12.47(s, 1H), 7.97(d, 1H),7.69(s, 1H), 369 7.24(t, 1H), 6.82-6.93(m, 5H), 6.48(t, 1H), 3.75(s,2H), 3.74(s, 3H), 3.27(q, 2H), 1.16(t, 3H) 16 (DMSO-d₆): 12.57(s, 1H),7.93(d, 1H), 7.72(s, 1H), 347 7.45(m, 1H), 6.96-7.25(m, 4H), 6.94(s,1H), 6.09(brs, 2H) 3.87(s, 2H), 17 (DMSO-d₆): 7.93(d, 1H), 7.67(s, 1H),7.08(s1H), 7.03(s, 1H), 389 6.94(d, 1H), 6.90(s, 1H), 6.06(s, 2H),5.94(s, 2H), 3.80(s, 2H) 20 (DMSO-d₆): 12.49(s, 1H), 7.99(d, 1H),7.71(s, 1H), 355 7.24(t, 1H), 6.82-6.94(m, 5H), 6.48(m, 1H), 3.75(s,2H), 3.74(s, 3H), 2.79(d, 3H) 21 (DMSO-d₆): 12.54(s, 1H), 7.98(d, 1H),7.71(s, 1H), 375 7.46(m, 1H), 7.21(m, 1H), 7.09(m, 1H), 6.93(d, 1H),6.91(s, 1H), 6.51(t, 1H), 3.87(s, 2H), 3.25(q, 2H), 1.15(t, 3H) 22(DMSO-d₆): 12.47(s, 1H), 7.98(d, 1H), 7.69(s, 1H), 417 7.08(s, 1H),(7.03, m, 1H), 6.89-6.97(m, 2H), 6.48(t, 1H), 6.05(s, 1H), 3.88(s, 2H),3.26(q, 2H), 1.16(t, 3H) 23 (DMSO-d₆): 12.56(s, 1H), 7.99(d, 1H),7.73(s, 1H), 361 7.45(m, 1H), 7.24(m, 1H), 7.09(m, 1H), 6.95(d, 1H),6.90(s, 1H), 6.50(m, 1H), 3.87(s, 2H), 2.79(d, 3H), 24 (DMSO-d₆):12.49(s, 1H), 7.92(d, 1H), 7.68(s, 1H), 403 7.24(t, 1H),6.85-6.94(m.5H), 5.95(s, 2H), 4.08(m, 2H), 3.75-3.82(m, 2H), 2.16(m, 2H)25 (CDCl₃): 9.28(brs, 1H), 8.09(d, 1H), 7.20-7.33(m, 7H), 4376.79-6.93(m, 5H), 4.51(d, 2H), 3.61(s, 2H), 3.75-3.82(m, 2H), 2.16(m,2H) 26 (CDCl₃): 8.85(brs, 1H), 8.08(d, 1H), 7.20-7.34(m, 6H), 4796.79-6.92(m, 5H), 5.99(s, 2H), 4.52(d, 2H), 3.66(s, 2H) 27(methanol-d₄): 7.90(d, 1H), 7.56(s, 1H), 7.39-7.43(m, 2H), 3457.29-7.31(m, 2H), 7.09-7.11(m, 2H), 3.99(s, 1H) 28 (methanol-d₄):7.91(d, 1H), 7.62(s, 1H), 7.39-7.41(m, 2H), 359 7.29-7.31(m, 2H),7.09-7.12(m, 2H), 3.99(s, 1H), 2.92(s, 3H) 29 (methanol-d₄): 7.91(d,1H), 7.55(s, 1H), 7.40(m, 2H), 373 7.30(m, 2H), 7.02-7.06(m, 2H),3.98(s, 2H), 3.32(m, 2H), 1.23(t, 3H) 30 (CDCl₃): 9.31(brs, 1H), 8.53(d,1H), 7.57(s, 1H), 7.47(d, 1H), 346 7.37(s, 1H), 7.34(m, 1H),6.88-6.95(m, 2H), 3.82(s, 2H), 2.61(s, 3H) 31 (CDCl₃): 9.15(brs, 1H),8.50(d, 1H), 7.53(s, 1H), 7.45(dd, 1H), 340 7.33(m, 2H), 6.91(m, 1H),6.86(m, 1H), 3.83(s, 3H), 3.80(s, 2H), 2.59(s, 3H) 32 (DMSO-d₆):12.36(brs, 1H), 8.47(d, 1H), 7.91(s, 1H), 388 7.71(s, 1H), 7.62(d, 1H),7.08(s, 1H), 7.04(s, 1H), 6.07(s, 2H), 3.88(s, 2H), 2.50(s, 3H) 33(DMSO-d₆): 12.62(s, 1H), 8.48(d, 1H), 7.92(s, 1H), 344 7.72(s, 1H),7.63(d, 1H), 7.45(m, 2H), 7.33(m, 2H), 4.00(s, 2H), 2.49(s, 3H) 34(methanol-d₄): 7.91(s, 1H), 7.82(d, 1H), 7.46(s, 1H), 327 7.35(dd, 1H),7.14(t, 1H), 6.81(m, 2H), 6.70(m, 1H), 3.73(2s, 2H) 35 (DMSO-d₆):12.55(s, 1H), 7.93(d, 1H), 7.69(s, 1H), 329 7.16-7.41(m, 4H),6.91-6.95(m, 2H), 5.94(m, 2H), 3.80(s, 2H) 36 (CDCl₃): 8.90(s, 1H),8.55(d, 1H), 7.60(s, 1H), 310 7.31-7.53(m, 6H), 3.806(s, 2H), 2.63(s,3H) 37 (DMSO-d₆): 9.36(s, 1H), 8.47(d, 1H), 7.91(s, 1H), 326 7.71(s,1H), 7.62(dd, 1H), 7.11(m, 2H), 6.64-6.76(m, 3H), 3.69(s, 2H), 2.50(s,3H) 38 (DMSO-d₆): 12.61(s, 1H), 8.48(d, 1H), 7.92(s, 1H), 328 7.72(s,1H), 7.64(m, 1H), 7.32-7.42(m, 2H), 7.17-7.21(m, 2H), 3.90(s, 2H),2.50(s, 3H) 39 (CDCl₃): 8.79(brs, 1H), 8.49(d, 1H), 7.52(s, 1H), 4667.43(dd, 1H), 7.31(m2H), 6.84-6.91(m, 3H), 4.03(t, 2H), 3.80(s, 2H),2.54(s, 3H), 2.49-2.65(brm, 8H), 2.27(s, 3H), 1.96(m, 2H) 40 (DMSO-d₆):12.55(s, 1H), 8.47(d, 1H), 7.91(s, 1H), 328 7.71(s, 1H), 7.62(dd, 1H),7.36(m, 2H), 7.15(m, 2H), 3.80(s, 2H), 2.50(s, 3H) 41 (DMSO-d₆):12.51(s, 1H), 7.92(d, 1H), 7.68(s, 1H), 311 7.24-7.34(m, 5H),6.91-6.95(m, 2H), 5.98(brs, 2H), 3.80(s, 2H) 42 (DMSO-d₆): 12.60(s, 1H),8.14(s, 1H), 7.96(d, 1H), 329 7.68(s, 1H), 7.30-7.39(m, 4H),7.14-7.19(m, 2H), 7.09(s, 1H), 6.99(s, 1H), 3.82(s, 2H) 43(methanol-d₄): 7.95(s, 1H), 7.81(d, 1H), 7.50(d, 1H), 404 7.37(dd, 1H),7.13-7.33(m, 4H), 3.81(s, 2H), 2.96(s, 3H) 44 (DMSO-d₆): 12.70(brs, 1H),9.80(brs, 1H), 8.47(d, 1H), 403 7.92(s, 1H), 7.71(s, 1H), 7.62(d, 1H),7.29(t, 1H), 7.19(s, 1H), 7.07-7.12(m, 2H), 3.78(s, 2H), 2.99(s, 3H) 45(methanol-d₄): 7.83(d, 1H), 7.54(s, 1H), 7.23(t, 1H), 467 7.09(m, 2H),6.92(m, 2H), 6.82(m, 1H), 4.03(t, 2H), 3.76(s, 2H), 2.56(brm, 10H),1.93-1.98(m, 2H) 46 (DMSO-d₆): 12.64(s, 1H), 8.46(d, 1H), 8.13(s, 1H),348 7.94(s, 1H), 7.86(dd, 1H), 7.32-7.42(m.2H), 7.17-7.21(m, 2H),3.90(s, 2H) 47 (DMSO-d₆): 12.64(s, 1H), 8.46(d, 1H), 8.13(s, 1H), 3667.94(s, 1H), 7.86(dd, 1H), 7.46(m, 1H), 7.24(m, 1H), 7.08(m, 1H),3.89(s, 2H) 48 (DMSO-d₆): 12.56(s, 1H), 9.35(s, 1H), 8.46(d, 1H), 3468.12(s, 1H), 7.94(s, 1H), 7.86(dd, 1H), 7.11(t, 1H), 6.74(m, 2H),6.65(dd, 1H), 3.70(s, 2H) 49 (DMSO-d₆): 12.60(brs, 1H), 8.45(d, 1H),8.12(s, 1H), 408 7.94(s, 1H), 7.86(dd, 1H), 7.08(s, 1H), 7.04(s, 1H),6.07(s, 2H), 3.89(s, 2H) 50 (DMSO-d₆): 12.60(s, 1H), 8.46(d, 1H),8.12(s, 1H), 330 7.94(s, 1H), 7.86(dd, 1H), 7.25-7.34(m, 5H), 3.81(s,2H) 51 (DMSO-d₆): 12.61(s, 1H), 9.73(s, 1H), 8.46(d, 1H), 423 8.12(s,1H), 7.94(s, 1H), 7.85(dd, 1H), 7.29(t, 1H), 7.19(s, 1H), 7.10(m, 2H),3.79(s, 2H), 2.99(s, 3H) 52 (methanol-d₄): 7.89(d, 2H), 7.74(s, 1H),7.23(t, 1H), 453 7.07(m, 2H), 6.81-6.92(m, 3H), 4.02(t, 2H), 3.75(s,2H), 2.84(brm, 4H), 2.47-2.52(brm, 6H), 1.95(m, 2H) 53 (DMSO-d₆):12.60(brs, 1H), 8.45(d, 1H), 8.12(s, 1H), 486 7.94(d, 1H), 7.86(dd, 1H),7.22(t, 1H), 6.83-6.91(m, 3H), 3.98(t, 2H), 3.75(s, 2H), 2.25-2.40(brm,10H), 2.12(s, 3H), 1.84(m, 2H) 54 (DMSO-d₆): 12.65(brs, 1H), 8.45(d,1H), 8.12(s, 1H), 500 7.94(s, 1H), 7.86(dd, 1H), 7.22(t, 1H),6.81-6.91(m, 3H), 3.98(t, 2H), 3.76(s, 2H), 2.20-2.45(brm, 12H), 1.86(m,2H), 0.95(t, 3H) 55 (DMSO-d₆): 8.45(d, 1H), 8.11(s, 1H), 7.93(s, 1H),472 7.85(dd, 1H), 7.22(t, 1H), 6.81-6.91(m, 3H), 3.98(t, 2H), 3.75(s,2H), 3.29(brs, 1H), 2.67(m, 4H), 2.37(m, 2H), 2.27(brm, 4H), 1.84(m, 2H)56 (DMSO-d₆): 12.65(s, 1H), 8.30(d, 1H), 8.11(s, 1H), 332 7.81(d, 1H),7.57(s, 1H), 7.32-7.42(m, 2H), 7.17-7.21(m, 2H), 3.90(s, 2H) 57(DMSO-d₆): 12.59(s, 1H), 8.28(d, 1H), 8.12(s, 1H), 332 7.80(d, 1H),7.56(s, 1H), 7.38(m, 2H), 7.16(t, 2H), 3.81(s, 2H) 58 (DMSO-d₆):12.65(s, 1H), 8.20(d, 1H), 8.11(s, 1H), 350 7.82(d, 1H), 7.57(s, 1H),7.45(m, 1H), 7.23(m, 1H), 7.07(m, 1H), 3.89(s, 2H) 59 (DMSO-d₆):12.61(s, 1H), 9.72(s, 1H), 8.28(d, 1H), 407 8.10(s, 1H), 7.80(m, 1H),7.56(s, 1H), 7.29(t, 1H), 7.12(s, 1H), 7.07-7.12(m, 2H), 3.79(s, 2H),2.99(s, 3H) 60 (methanol-d₄): 8.18(d, 1H), 7.80(s, 1H), 7.77(m, 1H), 4707.54(s, 1H), 7.23(t, 1H), 6.93(m, 2H), 6.82(dd, 1H), 4.04(t, 2H),3.76(s, 2H), 2.45-2.75(brm, 10H), 2.31(s, 3H), 1.94-2.00(m, 2H) 61(CDCl₃): 8.19(d, 1H), 8.0(s, 1H), 7.35(m, 2H), 7.02(s, 1H), 342.96.90(m, 2H), 6.84(d, 2H), 3.83(s, 3H), 3.79(s, 2H) 62 (CD₃CN): 9.50(s,1H), 8.34(d, 1H), 7.60(s, 1H), 7.52(s, 1H), 359 7.41(s, 1H), 7.29(m,1H), 7.06(s, 1H), 6.95(m, 2H), 6.85(m, 1H), 3.8(s, 3H), 3.70(s, 2H) 63(CD₃CN): 9.55(s, 1H), 8.5(d, 1H), 7.64(s, 1H), 7.56(s, 1H), 422 7.53(d,1H), 7.43(s, 1H), 7.36(t, 1H), 7.27(s, 1H), 7.18(m, 2H), 7.08(s, 1H),3.73(s, 2H), 2.97(s, 3H) 64 (CD₃CN): 9.63(s, 1H), 7.73(m, 1H), 7.50(m,1H), 7.29(m, 1H) 340 7.06(m, 3H), 6.94(m, 2H), 6.87(m, 1H), 3.81(s, 3H),3.71(s, 2H) 65 (CD₃CN): 9.60(s, 1H), 8.20(d, 1H), 7.60(s, 1H), 7.50(d,1H), 406 7.45(s, 1H), 7.37(m, 1H), 7.28(s, 1H), 7.22(s, 1H), 7.18(m,2H), 7.10(s, 1H), 3.69(s, 2H), 2.98(s, 3H) 66 (methanol-d₄): 8.14(d,1H), 7.54(m, 2H), 7.34(m, 3H), 313 7.25(m, 2H), 7.13(s, 1H), 3.74(s, 2H)68 (DMSO-d₆): 11.61(s, 1H), 9.67(s, 1H), 8.23(d, 1H), 424 7.74(d, 1H),7.60(d, 1H), 7.43(s, 1H), 7.14-7.24(m, 4H), 3.78(s, 2H), 2.93(s, 3H) 69(DMSO-d₆): 8.23(d, 1H), 7.74(d, 1H), 7.61(m, 1H), 7.44(m, 349 2H),7.20(m, 1H), 7.14(d, 1H), 7.07(m, 1H), 3.78(s, 2H) 70 (methanol-d₄):8.15(d, 1H), 7.82(d, 1H), 7.72(dd, 1H), 448 7.61(dd, 1H), 7.71-7.56(m,3H), 7.26(d, 1H), 7.14(d, 1H), 7.14(d, 1H), 3.83(s, 2H), 3.22(q, 4H),1.09(t, 6H) 71 (methanol-d₄): 8.15(d, 1H), 7.78(d, 1H), 7.69(m, 2H), 4627.63(m, 1H), 7.55(m, 2H), 7.28(d, 1H), 7.15(d, 1H), 3.87(s, 1H), 3.68(m,4H), 2.97(m, 4H) 72 (methanol-d₄): 8.1(d, 1H), 7.54(m, 2H), 7.27(s, 1H),454 7.23(t, 1H), 7.14(d, 1H), 6.91(m, 2H), 6.82(m, 1H), 3.99(t, 2H),3.69(s, 2H), 3.35(m, 2H), 2.95(m, 2H), 1.98(m, 2H), 1.80(m, 2H),1.65(brm, 1H), 1.48(m, 2H), 1.37(m, 2H) 73 (methanol-d₄): 8.14(d, 1H),7.52(m, 2H), 7.22-7.26(m, 2H), 455 7.13(d, 1H), 6.93(m, 2H), 6.84(dd,1H), 4.13(t, 2H), 3.69(s, 1H), 2.80(t, 2H), 2.40-2.60(brm, 8H), 2.26(s,3H) 74 (methanol-d₄): 8.15(d, 1H), 7.52(m, 2H), 7.23-7.27(m, 2H), 4697.14(d, 1H), 6.92(m, 2H), 6.84(dd, 1H), 4.11(t, 2H), 3.70(s, 1H),3.30-3.55(brm, 8H), 3.20(m, 2H), 2.91(s, 3H), 2.16(m, 2H) 75 (DMSO-d₆):11.4(brs, 1H), 7.87(d, 1H), 7.30(s, 1H), 403 6.92(m, 2H), 6.72-6.80(m,3H), 6.61(s, 1H), 6.49) (d, 1H), 5.86(s, 2H), 3.48(s, 2H), 2.57(s, 3H)76 (DMSO-d₆): 11.49(brs, 1H), 9.70(brs, 1H), 7.89(d, 1H), 421 7.35(s,1H), 7.13-7.34(m, 3H), 6.97(d, 1H), 6.73(dd, 1H), 6.61(s, 1H), 5.88(s,2H), 3.74(s, 2H), 2.94(s, 3H) 77 (methanol-d₄): 7.85(d, 1H), 7.30(d,1H), 7.20(t, 1H), 451 7.05(d, 1H), 6.77-6.90(m, 5H), 3.96(t, 2H),3.67(s, 2H), 3.02(brm, 2H), 2.57(m, 2H), 1.71-1.79(m, 4H), 1.40(m, 2H),1.14(m, 2H) 78 (methanol-d₄): 8.15(d, 1H), 7.79(s, 1H), 7.70(d, 1H), 4207.66(d, 1H), 7.60(t, 1H), 7.53(m, 2H), 7.27(s, 1H), 7.14(s, 1H), 3.86(s,1H), 2.68(s, 6H) 79 (methanol-d₄): 7.78(m, 2H), 7, 70(m, 1H), 7.65(m,2H), 417 7.60(m, 1H), 7.18(d, 1H), 7.15(d, 2H), 3.87(s, 2H), 2.67(s, 6H)80 (methanol-d₄): 8.53(d, 1H), 8.08(s, 1H), 8.02(d, 1H), 338.9 7.9(s,1H) 7.28(s, 1H), 7.23(m, 1H), 6.94(m, 2H), 6.83(d, 1H), 3.78(s, 3H),3.7(s, 2H), 2.75(s, 3H) 81 (methanol-d₄): 8.56(d, 1H), 8.08(s, 1H),7.99(d, 1H), 326.9 7.87(s, 1H), 7.47(m, 1H), 7.38(m, 2H), 7.15(m, 2H),3.83(s, 2H), 2.75(s, 3H) 82 (methanol-d₄): 8.53(d, 1H), 8.08(s, 1H),8.00(d, 1H), 402 7.9(s, 1H), 7.28(m, 3H), 7.12(m, 2H), 3.75(s, 2H),2.95(s, 3H), 2.75(s, 3H) 83 (methanol-d₄): 8.53(d, 1H), 8.08(s, 1H),8.02(d, 1H), 416 7.9(s, 1H), 7.78(s, 1H), 7.67(m, 2H), 7.59(m, 1H),7.30(s, 1H), 3.88(s, 2H), 2.78(s, 3H), 2.67(s, 6H) 84 (methanol-d₄):8.53(d, 1H), 8.10(s, 1H), 8.02(d, 1H), 431 7.92(s, 1H), 7.26(m, 3H),7.12(d, 1H), 7.05(d, 1H), 3.73(s, 2H), 2.77(s, 3H), 2.72(s, 6H) 85(methanol-d₄): 8.53(d, 1H), 8.05(s, 1H), 7.98(d, 1H), 324.9 7.88(s, 1H),7.27(s, 1H), 7.12(m, 1H), 6.77(m, 2H), 6.68(d, 1H), 3.65(s, 2H), 2.75(s,3H) 86 (CD₃CN): 9.01(s, 1H), 8.53(d, 1H), 7.9(s, 1H), 7.88(d, 1H), 4207.72(s, 1H), 7.60(s, 1H), 7.32(m, 1H), 7.25(m, 1H), 7.20(s, 1H), 3.81(s,2H), 2.97(s, 3H), 2.74(s, 3H) 87 (CD₃CN): 9.76(s, 1H), 8.56(d, 1H),7.85(s, 1H), 7.82(d, 1H), 308.9 7.64(s, 1H), 7.34(m, 3H), 7.27(m, 2H),7.15(s, 1H), 3.73(s, 2H), 2.73(s, 3H) 88 (methanol-d₄): 8.15(d, 1H),7.56(d, 2H), 7.29(s, 1H), 328.9 7.15(m, 2H), 6.8(d, 2H), 6.69(m, 1H),3.63(s, 2H) 89 (DMSO-d₆): 11.63(s, 1H), 8.7(bs, 1H), 8.66(d, 1H), 450.957.93(m, 3H), 7.24(m, 2H), 6.92(m, 2H), 6.85(m, 1H), 4.0(t, 2H), 3.68(s,2H), 3.22(bs, 4H), 2.90(bs, 4H), 2.65(s, 3H), 2.1(s, 2H), 2.0(bs, 2H) 90(DMSO-d₆): 11.63(s, 1H), 8.70(d, 1H), 8.10(s, 1H), 8.0(s, 2H), 464.97.25(t, 2H), 6.92(m, 2H), 6.85(m, 1H), 4.0(t, 2H), 3.70(s, 2H), 3.22(bs,4H), 2.90(bs, 4H), 2.77(s, 3H), 2.70(s, 3H), 2.1(s, 2H), 2.0(bs, 2H) 91(DMSO-d₆): 11.67(s, 1H), 9.05(bs, 1H), 8.70(d, 1H), 550.0 8.1(s, 1H),8.05(m, 1H), 7.28(m, 2H), 6.95(m, 2H), 6.85(d, 1H), 4.07(t, 2H), 3.85(s,2H), 3.52(d, 2H), 3.23(m, 2H), 2.92(m, 2H), 2.82(s, 3H), 2.15(m, 2H),1.85(m, 2H), 1.67(m, 3H), 1.41(m, 1H) 92 (DMSO-d₆): 11.63 s 91H),8.69(d, 1H), 8.60(bs, 1H), 436.9 7.95(m, 3H), 7.26(m, 2H), 6.95(d, 2H),6.88(d, 1H), 4.12(t, 2H), 3.71(d, 2H), 3.19(bs, 4H), 2.92(m, 4H),2.69(s, 3H), 2.11(s, 2H) 93 (DMSO-d₆): 11.62(s, 1H), 8.65(d, 1H),7.95(m, 3H), 450.95 7.26(m, 2H), 6.93(d, 2H), 6.85(d, 1H), 4.1(bs, 2H),3.7(s, 2H), 3.38(m, 2H), 3.15(m, 2H), 3.0(m, 1H), 2.85(bs, 1H), 2.76(bs,2H), 2.66(s, 3H), 2.07(s, 2H) 94 (DMSO-d₆): 11.67(s, 1H), 9.31(bs, 1H),8.7(d, 1H), 8.1(s, 1H), 435.9 8.0(m, 2H), 7.3(t, 1H), 7.24(s, 1H),6.96(m, 2H), 6.9(m, 1H), 4.32(t, 2H), 3.85(s, 2H), 3.52(m, 4H), 3.03(m,2H), 2.72(s, 3H), 1.83(m, 2H), 1.70(m, 3H), 1.4(m, 1H), 107(methanol-d₄): 7.86(d, 1H), 7.34(s, 1H), 7.25(t, 1H), 452.1 7.07(s, 1H),6.94(m, 2H), 6.86(m, 2H), 6.80 s 91H), 4.16(t, 2H), 3.70(s, 2H), 2.88(t,2H), 2.73(bs, 8H), 2.45(s, 3H)

Example 4 ROCK Inhibition Assay

Compounds were screened for their ability to inhibit ROCK I (AA 6-553)activity using a standard coupled enzyme system (Fox et al. Protein Sci.7:2249, 1998). Reactions were carried out in a solution containing 100mM HEPES (pH 7.5), 10 mM MgCl₂, 25 mM NaCl, 2 mM DTT and 1.5% DMSO.Final substrate concentrations in the assay were 45 μM ATP (SigmaChemicals, St Louis, Mo.) and 200 μM peptide (American Peptide,Sunnyvale, Calif.). Reactions were carried out at 30° C. and 45 nM ROCKI. Final concentrations of the components of the coupled enzyme systemwere 2.5 mM phosphoenolpyruvate, 350 μM NADH, 30 μg/ml pyruvate kinaseand 10 μg/ml lactate dehydrogenase. Results are shown in Table 2, wherea K_(i) of less than 0.1 μM is defined as “A,” a K_(i) of between 0.1 μMand 1.0 μM is defined as “B,” and a K_(i) of greater than 1.0 μM isdefined as “C.” TABLE 2 Compound ROCK No. K_(i) 1 B 2 B 3 C 4 C 5 C 6 C7 C 8 C 9 C 10 B 11 C 12 C 13 B 14 C 15 B 16 A 17 A 20 B 21 B 22 B 23 B24 B 25 B 26 B 27 A 28 B 29 B 30 B 31 B 32 A 33 B 34 A 35 A 36 B 37 A 38B 39 B 40 B 41 A 42 B 43 B 44 B 45 A 46 B 47 B 48 A 49 A 50 B 51 B 52 A53 A 54 A 55 A 56 A 57 B 58 A 59 A 60 A 61 A 62 A 63 A 64 A 65 A 66 A 67A 68 A 69 A 70 A 71 B 72 A 73 A 74 A 75 A 76 A 77 A 78 B 79 B 80 B 81 B82 B 83 C 84 B 85 A 86 A 87 B 88 A 89 A 90 B 91 C 92 B 93 B 94 C 95 A 96A 97 A 98 A 99 A 100 A 101 A 102 A 103 A 104 B 105 A 106 A 107 A

Example 5 Cytochrome P450 Inhibition

Selected compounds of the invention were assayed for their ability toinhibit various cytochrome P450 isozymes, such as, for example CYP2D6,CYP2C9, CYP1A2, CYP2C19, CYP3A4, CYP2C9, and CYP3A4 (human livermicrosome). Such assays are described by Crespi et al., Anal. Biochem.248:188-90, 1997; Ekins et al., J. Pharmacol. Exp. Ther. 290, 429-38,1999; and Ekins et al., J. Pharmacol. Exp. Ther. 291, 424-33, 1999. In atypical isozyme inhibition experiment, test compounds are placed into amicrowell on a microtiter plate as an 8 nL solution (2 mM in 75%DMSO/water). The plate is centrifuged for 2 minutes at approximately1000 rpm to shift the compound to the bottom of the well.Polyvinylpyrrolidone (PVP/10K, 100 mL, 0.2% in 75% DMSO/water) is addedto the well and this excipient also centrifuged for 2 minutes atapproximately 1000 rpm to ensure mixing with the compound. The platescontaining the wells are dried in vacuo. Distilled water (800 nL) isadded to the wells containing compound, the background control wells,and the positive control wells, followed by the addition of 200 mL ofphosphate buffer (500 mM potassium phosphate butter at pH=8.4). Thepositive control cells contain a drug such as, for example, miconazole.Insect baculosomes (PanVera P2315) in 500 nM phosphate buffer was addedto the background control wells and the plate was scanned for compoundfluorescence. NADP⁺ (200 mL, 100 μM) and substrate (3A4:5 μM Vivid™ 3A4Red, 2C9:1 μM Vivid™ 2C9 Green, 1A2:2 μM Vivid™ 1A2 Blue, 2C19:10 μMVivid™ 2C19 Blue, and 2D6:10 μM Vivid™ 2D6 Blue) in 100 mM phosphatebuffer was added to each cell at a final concentration corresponding tothe K_(m) of the substrate for its pertinent CYP450 isozyme. Therefore,CYP450 isozyme (400 mL) and recycling buffer (3.3 mMglucose-6-phosphate, 0.4 units/mL glucose-6-phosphate dehydrogenase, 100mM MgCl₂, and 0.00025% Antifoam 289) in 100 mM phosphate buffer wasadded to each well containing compound and background control to obtainthe following concentrations: 5 nM CYP3A4, 10 nM CYP2C9, 5 nM CYP1A2, 5nM CYP2C19, and 20 nM CYP2D6. After incubation for 60 minutes, the wellswere read with a fluorescent plate reader. Cytochrome P450 isozymeinhibition values are given in Table 3, where an IC₅₀ of less than 1.0μM is defined as “A,” an IC₅₀ of between 1.0 μM and 10.0 μM is definedas “B,” and an IC₅₀ of greater than 10.0 μM is defined as “C.” TABLE 3Compound CYP1A2 CYP2C19 CYP2C9 CYP2D6 CYP3A4 CYP3A4 (HLM) No. K_(i)K_(i) K_(i) K_(i) K_(i) K_(i) 61 C B B C B B 62 C — B — B B 64 B C B B BC 72 C B — B B C 73 C — C C C C 74 C C C C C C 77 C B B A B B 80 C B B CB B 85 C B C C C C 89 C B C — C C 90 C — C — C C 91 C C C — C C 92 C C C— C C 93 C C C — C C 94 C C C — C C 101 B B B — B B 102 C B C C B C 103C C C C C C 105 B A B A B B 107 C C C A — C

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A compound having the formula:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is halogen,—CN, —NO₂, —NR₂, —OR, —SR, or an optionally substituted C₁₋₄ aliphaticor C₃₋₆ cycloaliphatic; R² is hydrogen or C₁₋₃ aliphatic; each R³ is,independently, hydrogen, halogen, —NR₂, —OR, —SR, or an optionallysubstituted C₁₋₄ aliphatic group or C₃₋₆ cycloaliphatic group, or twoR³, taken together with the intervening carbon atom, form an optionallysubstituted 3-6 membered cycloaliphatic or heterocyclyl ring having 1-2heteroatoms selected from nitrogen, oxygen, or sulfur; X¹ is CR⁴ or N,wherein R⁴ is hydrogen, halogen, —C(O)R, —C(S)R, —C(O)N(R)₂, —C(S)N(R)₂,—CN, —NO₂, or an optionally substituted C₁₋₄ aliphatic group; A¹ is aphenyl ring or a 5-6 membered heteroaryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, wherein saidphenyl or heteroaryl ring is optionally substituted with 1-5 independentoccurrences of TR⁵, wherein two TR⁵ groups are optionally taken togetherto form methylenedioxy or ethylenedioxy; T is a bond or a C₁-C₆alkylidene chain, wherein up to two methylene units of T are optionallyand independently replaced by —NR′—, —S—, —O—, —C(S)—, —C(O)O—, —OC(O)—,—C(O)—, —C(O)C(O)—, —C(O)NR′—, —NR′C(O)—, —NR′C(O)O—, —S(O)₂NR′—,—NR′S(O)₂—, —C(O)NR′NR′—, —NR′C(O)NR′—, —OC(O)NR′—, —NR′NR′—,—NR′S(O)₂NR′—, —S(O)—, —S(O)₂—, —P(O)—, —P(O)₂—, —P(O)R′; each R⁵ is,independently, R^(a), halogen, —NO₂, or —CN; each occurrence of R^(a)is, independently, hydrogen, an optionally substituted moiety selectedfrom a C₁₋₄ aliphatic group, a C₃₋₆ cycloaliphatic group, a C₁₋₄haloaliphatic group, a 6-10 membered mono- or bicyclic aryl ring, a 3-10membered mono- or bicyclic cycloaliphatic ring, or a 5-14 membered mono-or bicyclic heteroaryl or heterocyclyl ring having 1-5 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or whereinR^(a), R′, and an intervening nitrogen atom together form an optionallysubstituted 5-6 membered heterocyclic or heteroaryl ring optionallyhaving 1-2 additional heteroatoms selected from nitrogen, oxygen, orsulfur; each optionally substituted group or ring is, independently,optionally substituted with 1-5 substituents independently selected fromC₁₋₄ aliphatic, C₃₋₆ cycloaliphatic, C₁₋₄ haloaliphatic, halogen, —OR″,—OC(O)N(R″)₂, —OC(O)R″, —OC(O)OR″, —NO₂, —N(R″)₂, —NR″C(O)R″,—NR″C(S)R″, —NR″C(O)N(R″)₂, —NR″C(S)N(R″)₂, —NR″C(O)OR″, —NR″NR″C(O)R″,—NR″NR″C(O)N(R″)₂, —NR″NR″C(O)OR″, —NR″S(O)₂N(R″)₂, —NR″S(O)₂R″,—N(OR″)R″, —CN, C(O)OR″, —C(O)R″, —C(S)R″, —C(O)N(R″)₂, —C(S)N(R″)₂,—OC(O)N(R″)₂, —OC(O)R″, —C(O)N(OR″)R″, —C(═NOR″)R″, —C(═NH)—N(R″)₂,—SR″, —SC(O)R″, —SC(S)R″, —S(O)R″, —S(O)₂R″, —S(O)₂OR″, or —S(O)₂N(R″)₂;each occurrence of R is, independently, hydrogen, a C₁₋₄ aliphaticgroup, a C₃₋₆ cycloaliphatic group, —(CH₂)₁₋₂Ph, —CH═CHPh, or a C₁₋₄haloaliphatic group, or two R groups and an intervening nitrogen atomtogether form an optionally substituted 5-6 membered heterocyclic orheteroaryl ring optionally having 1-2 additional heteroatoms selectedfrom nitrogen, oxygen, or sulfur; each occurrence of R′ is,independently, hydrogen, a C₁₋₄ aliphatic group, a C₃₋₆ cycloaliphaticgroup, —(CH₂)₁₋₂Ph, —CH═CHPh, or a C₁₋₄ haloaliphatic group, or two R′groups and an intervening nitrogen atom together form an optionallysubstituted 5-6 membered heterocyclic or heteroaryl ring optionallyhaving 1-2 additional heteroatoms selected from nitrogen, oxygen, orsulfur; and each occurrence of R″ is, independently, hydrogen, a C₁₋₄aliphatic group, a C₃₋₆ cycloaliphatic group, —(CH₂)₁₋₂Ph, —CH═CHPh, ora C₁₋₄ haloaliphatic group, or two R″ groups and an intervening nitrogenatom together form an optionally substituted 5-6 membered heterocyclicor heteroaryl ring optionally having 1-2 additional heteroatoms selectedfrom nitrogen, oxygen, or sulfur.
 2. The compound according to claim 1or a pharmaceutically acceptable salt thereof, wherein R¹ is halogen,—NR₂, —OR, or an optionally substituted C₁₋₄ aliphatic.
 3. The compoundaccording to claim 2 or a pharmaceutically acceptable salt thereof,wherein R¹ is C₁₋₄ aliphatic.
 4. The compound according to claim 3 or apharmaceutically acceptable salt thereof, wherein R¹ is CH₃ or CH₂CH₃.5. The compound according to claim 2 or a pharmaceutically acceptablesalt thereof, wherein R¹ is halogen.
 6. The compound according to claim5 or a pharmaceutically acceptable salt thereof, wherein said halogen isfluoro or chloro.
 7. The compound according to claim 2 or apharmaceutically acceptable salt thereof, wherein R¹ is NR₂.
 8. Thecompound according to claim 7 or a pharmaceutically acceptable saltthereof, wherein R¹ is NH₂, NHCH₃, N(CH₃)₂, NHCH₂CH₃, or NHCH₂Ph.
 9. Thecompound according to claim 2 or a pharmaceutically acceptable saltthereof, wherein R¹ is OR.
 10. The compound according to claim 9 or apharmaceutically acceptable salt thereof, wherein R¹ is OCH₃ or OCH₂CH₃.11. The compound according to claim 1, wherein R² is hydrogen.
 12. Thecompound according to claim 1, wherein each R³ is, independently,selected from hydrogen, halogen, —NR′″₂, —OR′″, —SR′″, or a C₁₋₄aliphatic group, wherein each R′″ is, independently, hydrogen or a C₁₋₄aliphatic group.
 13. The compound according to claim 12, wherein R³ is,independently, selected from hydrogen, chloro, fluoro, or —OH.
 14. Thecompound according to claim 12, wherein each R³ is hydrogen.
 15. Thecompound according to claim 1, wherein A¹ is a phenyl ring or a 5-6membered heteroaryl ring having 1-2 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, wherein said phenyl or heteroaryl ringis optionally substituted with 1-3 independent occurrences of TR⁵,wherein two TR⁵ groups are optionally taken together to formmethylenedioxy or ethylenedioxy.
 16. The compound according to claim 15,wherein A¹ is a phenyl ring, optionally substituted with 1-3 occurrencesof TR⁵.
 17. The compound according to claim 16, wherein one of −TR⁵ isat the 3-position.
 18. The compound according to claim 17, wherein −TR⁵is chloro, fluoro, —OH, optionally substituted C₁₋₄ alkoxy,—NHS(O)₂R^(a), —S(O)₂NRR^(a).
 19. The compound according to claim 1,wherein X¹ is CR⁴, wherein R⁴ is hydrogen or a C₁₋₄ aliphatic group. 20.The compound according to claim 19, wherein, X¹ is CH.
 21. The compoundaccording to claim 1, wherein X¹ is N.
 22. The compound according toclaim 1, having the formula:

R¹ is C₁₋₄ aliphatic, halogen, —NR₂, —OR, or —SR; each T is,independently, a bond or a C₁-C₆ alkylidene chain, wherein up to twomethylene units of T are optionally and independently replaced by —NR′—,—S—, —O—, —C(O)NR′—, —NR′C(O)—, —NR′C(O)O—, —S(O)₂NR′—, —NR′S(O)₂—,—NR′C(O)NR′—, —OC(O)NR′—, or —NR′S(O)₂NR′—, or two TR⁵ groups areoptionally taken together to form methylenedioxy or ethylenedioxy; eachR⁵ is, independently, R^(a) or halogen; and n is 1 to
 3. 23-38.(canceled)
 39. A compound selected from the group of compounds listed inTable
 1. 40. The compound according to claim 1, wherein said compoundbinds to ROCK with a K_(i) of less than or equal to 0.1 micromolar andinhibits at least one of the cytochrome P450 isozymes selected from thegroup consisting of CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4 with anIC₅₀ of greater than or equal to 5 micromolar.
 41. The compoundaccording to claim 40, wherein said compound inhibits at least 3 of thecytochrome P450 isozymes selected from the group consisting of CYP1A2,CYP2C19, CYP2C9, CYP2D6, and CYP3A4 with an IC₅₀ of greater than orequal to 5 micromolar.
 42. The compound according to claim 41, whereinsaid compound inhibits at least 4 of the cytochrome P450 isozymesselected from the group consisting of CYP1A2, CYP2C19, CYP2C9, CYP2D6,and CYP3A4 with an IC₅₀ of greater than or equal to 5 micromolar.
 43. Acomposition comprising a compound according to claim 1 and apharmaceutically acceptable carrier, adjuvant, or vehicle.
 44. A methodof inhibiting ROCK kinase activity in a biological sample comprisingcontacting said biological sample with a compound according to claim 1,or a pharmaceutical composition thereof.
 45. The composition accordingto claim 43, said composition comprising an additional therapeutic agentselected from a chemotherapeutic or anti-proliferative agent, ananti-inflammatory agent, an immunomodulatory or immunosuppressive agent,a neurotrophic factor, an agent for treating cardiovascular disease, anagent for treating destructive bone disorders, an agent for treatingliver disease, an anti-viral agent, an agent for treating blooddisorders, an agent for treating diabetes, or an agent for treatingimmunodeficiency disorders.
 46. A method of treating or lessening theseverity of a disease, condition, or disorder in a patient selected fromangina, atherosclerosis, cerebral vasospasm, cerebrovascularcontraction, coronary vasospasm, endothelial dysfunction, erectiledysfunction, glaucoma, hypertension, ischemic/reperfusion injury,myocardial hypertrophy, myocardial infarction, peripheral circulationdisorder, pre-term labor, Raynaud's Disease, renal disease, or strokecomprising the step of administering to said patient a therapeuticallyeffective dose of a composition according to claim
 43. 47. The method ofclaim 46, wherein said cardiovascular disease, conditions, or disorderis glaucoma. 48-50. (canceled)
 51. The method of claim 46, comprisingthe additional step of administering to said patient an additionaltherapeutic agent selected from a chemotherapeutic or anti-proliferativeagent, an anti-inflammatory agent, an immunomodulatory orimmunosuppressive agent, a neurotrophic factor, an agent for treatingcardiovascular disease, an agent for treating destructive bonedisorders, an agent for treating blood disorders, an agent for treatingdiabetes, or an agent for treating immunodeficiency disorders, whereinsaid additional therapeutic agent is appropriate for the disease beingtreated and said additional therapeutic agent is administered togetherwith said composition as a single dosage form or separately from saidcomposition as part of a multiple dosage form.